@article {70264, title = {Live dynamics of induced cell-cell fusion between mitotic and interphasic cells}, journal = {bioRxiv}, year = {2026}, month = {2026 Jan 27}, abstract = {

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed an unexpected plasticity in cell cycle control and highlight cell-cell fusion experiments as a powerful experimental system to study how competing cytoplasmic states are integrated in a shared cytoplasm.

}, doi = {10.64898/2026.01.27.700572}, url = {https://www.biorxiv.org/content/early/2026/01/27/2026.01.27.700572}, author = {Afonso, Olga and Feliciano, Daniel and Lippincott-Schwartz, Jennifer} } @article {70281, title = {Live dynamics of induced cell-cell fusion between mitotic and interphasic cells}, journal = {bioRxiv}, year = {2026}, month = {2026 Jan 27}, abstract = {

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed an unexpected plasticity in cell cycle control and highlight cell-cell fusion experiments as a powerful experimental system to study how competing cytoplasmic states are integrated in a shared cytoplasm.

}, doi = {10.64898/2026.01.27.700572}, url = {https://www.biorxiv.org/content/early/2026/01/27/2026.01.27.700572}, author = {Afonso, Olga and Feliciano, Daniel and Lippincott-Schwartz, Jennifer} } @article {70282, title = {Live dynamics of induced cell-cell fusion between mitotic and interphasic cells}, journal = {bioRxiv}, year = {2026}, month = {2026 Jan 27}, abstract = {

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed an unexpected plasticity in cell cycle control and highlight cell-cell fusion experiments as a powerful experimental system to study how competing cytoplasmic states are integrated in a shared cytoplasm.

}, doi = {10.64898/2026.01.27.700572}, url = {https://www.biorxiv.org/content/early/2026/01/27/2026.01.27.700572}, author = {Afonso, Olga and Feliciano, Daniel and Lippincott-Schwartz, Jennifer} } @article {70242, title = {mRNAbow: A versatile gene expression system for multiplexed fluorescent imaging using optimized in vitro transcribed mRNA}, journal = {bioRxiv}, year = {2026}, month = {2026 Jan 02}, abstract = {

Messenger RNA (mRNA) transfection enables rapid, transient protein expression without nuclear entry, providing a powerful alternative to DNA or viral delivery in post-mitotic and otherwise difficult-to-transfect cells. Although in vitro transcribed (IVT) mRNAs have revolutionized therapeutic applications, their adoption in experimental biology remains limited by challenges in synthesis, variability across cell types, and concerns about cytotoxicity. Here, we define design principles that maximize IVT mRNA performance across diverse cellular and organismal systems. Through systematic comparison of capping strategies and base modifications, including N1-methyl-pseudouridine, 5-methylcytidine, and 5-methoxyuridine, we identify modifications that enhance translation while minimizing activation of cellular stress responses. Optimized transcripts drive robust protein expression within four hours, persist for up to one week, and support multiplexed expression of structurally and functionally distinct proteins in mammalian cells, including cancer cell lines, iPSC-derived systems, primary cells, and organoids, as well as in vivo in zebrafish embryos and in less genetically tractable models such as Danionella cerebrum and sea urchin embryos. To further expand accessibility for community use, we developed mRNAbow, a platform for generating low-toxicity mRNAs encoding organelle-targeted fluorescent proteins and biosensors for multiplex imaging, with corresponding plasmids made publicly available. Together, these advances establish a generalizable framework for IVT mRNA design and expand experimental access to synthetic mRNA technologies for dissecting cellular architecture and dynamics.

}, doi = {10.64898/2026.01.02.697412}, url = {https://www.biorxiv.org/content/early/2026/01/02/2026.01.02.697412}, author = {Choi, Heejun and Halanych, Coral and Kasberg, William and Testa, Michael D. and Rubin-Elgressy, Sarah and Nguyen, Phuong and Walpita, Deepika and Tsang, Arthur and Cortes, Dan and Song, Erin Yue and Wu, Haodi and Weissman, Irving L and Espinosa-Medina, Isabel and Satou, Chie and Song, Jia L. and Matus, David Q. and Lippincott-Schwartz, Jennifer} } @article {70179, title = {Secretome translation shaped by lysosomes and lunapark-marked ER junctions.}, journal = {Nature}, year = {2026}, month = {2026 Jan 01}, pages = {227{\textendash}236 }, abstract = {

The endoplasmic reticulum (ER) is a highly interconnected membrane network that serves as a central site for protein synthesis and maturation. A crucial subset of ER-associated transcripts, termed secretome mRNAs, encode secretory, lumenal and integral membrane proteins, representing nearly one-third of human protein-coding genes. Unlike cytosolic mRNAs, secretome mRNAs undergo co-translational translocation, and thus require precise coordination between translation and protein insertion. Disruption of this process, such as through altered elongation rates, activates stress response pathways that impede cellular growth, raising the question of whether secretome translation is spatially organized to ensure fidelity. Here, using live-cell single-molecule imaging, we demonstrate that secretome mRNA translation is preferentially localized to ER junctions that are enriched with the structural protein lunapark and in close proximity to lysosomes. Lunapark depletion reduced ribosome density and translation efficiency of secretome mRNAs near lysosomes, an effect that was dependent on eIF2-mediated initiation and was reversed by the integrated stress response inhibitor ISRIB. Lysosome-associated translation was further modulated by nutrient status: amino acid deprivation enhanced lysosome-proximal\ translation, whereas lysosomal pH neutralization suppressed it. These findings identify a mechanism by which ER junctional proteins and lysosomal activity cooperatively pattern secretome mRNA translation, linking ER architecture and nutrient sensing to the production of secretory and membrane proteins.

}, issn = {1476-4687}, doi = {10.1038/s41586-025-09718-0}, url = {https://doi.org/10.1038/s41586-025-09718-0}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J and Grimm, Jonathan and Wang, Nan and Lavis, Luke D and Singer, Robert H and Lippincott-Schwartz, Jennifer} } @article {70171, title = {Design principles of ciliary signaling.}, journal = {J Cell Sci}, volume = {138}, year = {2025}, month = {2025 Oct 15}, abstract = {

Primary cilia are microtubule-based sensory organelles that have been conserved throughout eukaryotic evolution. As discussed in this Review, a cilium is an elongated and highly specialized structure, and, together with its ability to selectively traffic and concentrate proteins, lipids and second messengers, it creates a signaling environment distinct from the cell body. Ciliary signaling pathways adopt a bow-tie network architecture, in which diverse inputs converge on shared effectors and second messengers before diverging to multiple outputs. Unlike other cellular bow-tie systems, cells exploit ciliary geometry, compartmentalization and infrastructure to enhance sensitivity at multiple scales, from individual molecular reactions to entire signaling pathways. In cilia, integration of the bow-tie network architecture with their specialized structure and unique environment confers robustness and evolvability, which enables cilia to acquire diverse signaling roles. However, this versatility comes with vulnerability - rare mutations that disrupt the features most essential for cilia robustness cause multisystem ciliopathies.

}, keywords = {Animals, Cilia, Ciliopathies, Humans, Signal Transduction}, issn = {1477-9137}, doi = {10.1242/jcs.264325}, url = {https://doi.org/10.1242/jcs.264325}, author = {Ott, Carolyn M and Lippincott-Schwartz, Jennifer} } @article {70178, title = {ER-to-Golgi trafficking is a nutrient-sensitive checkpoint linking glucose starvation to cell surface remodeling}, journal = {bioRxiv}, year = {2025}, month = {2025 October 31}, abstract = {

Cancer cells adapt to nutrient stress by remodeling the repertoire of proteins on their surface, enabling survival and progression under starvation conditions. However, the molecular mechanisms by which nutrient cues reshape the cell surface proteome to influence cell behavior remain largely unresolved. Here, we show that acute glucose starvation, but not amino acid deprivation or mTOR inhibition, selectively impairs ER-to-Golgi export of specific cargoes, such as E-cadherin, in a SEC24C-dependent manner. Quantitative cell surface proteomics reveal that glucose deprivation remodels the cell surface proteome, notably reducing surface expression of key adhesion molecules. This nutrient-sensitive reprogramming enhances cell migration in vitro and promotes metastasis in vivo. Mechanistically, we show that AMPK and ULK1 signaling orchestrate this process independent of autophagy, with ULK1-mediated phosphorylation of SEC31A driving SEC24C-dependent COPII reorganization. These findings establish ER-to-Golgi trafficking as a nutrient-sensitive regulatory node that links metabolic stress to cell surface remodeling and metastatic potential.

}, doi = {10.1101/2025.10.31.685804}, url = {https://www.biorxiv.org/content/early/2025/11/01/2025.10.31.685804}, author = {Joo, Joung Hyuck and Kasberg, William and Douglas, Spencer and Udoh, Uwemedimo and Carisey, Alexandre and Messing, James and Wang, Yong-Dong and Narina, Shilpa and Pruett-Miller, Shondra M. and Labelle, Myriam and Lippincott-Schwartz, Jennifer and Chang, Chi-Lun and Kundu, Mondira} } @article {69865, title = {FilaBuster: A Strategy for Rapid, Specific, and Spatiotemporally Controlled Intermediate Filament Disassembly}, journal = {bioRxiv}, year = {2025}, month = {2025 Apr 20}, abstract = {

Intermediate filaments (IFs) play key roles in cellular mechanics, signaling, and organization, but tools for their rapid, selective disassembly remain limited. Here, we introduce FilaBuster, a photochemical approach for efficient and spatiotemporally controlled IF disassembly in living cells. FilaBuster uses a three-step strategy: (1) targeting HaloTag to IFs, (2) labeling with a covalent photosensitizer ligand, and (3) light-induced generation of localized reactive oxygen species to trigger filament disassembly. This modular strategy applies broadly across IF subtypes{\textemdash}including vimentin, GFAP, desmin, peripherin, and keratin 18{\textemdash}and is compatible with diverse dyes and imaging platforms. Using vimentin IFs as a model system, we establish a baseline implementation in which vimentin-HaloTag labeled with a photosensitizer HaloTag ligand triggers rapid and specific IF disassembly upon light activation. We then refine this approach by (i) expanding targeting strategies to include a vimentin nanobody-HaloTag fusion, (ii) broadening the range of effective photosensitizers, and (iii) optimizing irradiation parameters to enable precise spatial control over filament disassembly. Together, these findings position FilaBuster as a robust platform for acute, selective, and spatiotemporally precise disassembly of IF networks, enabling new investigations into their structural and functional roles in cell physiology and disease.

}, doi = {10.1101/2025.04.20.649718}, url = {https://www.biorxiv.org/content/early/2025/04/21/2025.04.20.649718}, author = {Moore, Andrew S. and Krug, Tommy and Hansen, Simon B. and Ludlow, Alexander V. and Grimm, Jonathan B. and Ayala, Anthony X. and Plutkis, Sarah E. and Wang, Nan and Goldman, Robert D. and Medalia, Ohad and Lavis, Luke D. and Weitz, David A. and Lippincott-Schwartz, Jennifer} } @article {69945, title = {Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 May 27}, pages = {e2426644122}, abstract = {

Calreticulin (CALR) is primarily an endoplasmic reticulum chaperone protein that also plays a key role in facilitating programmed cell removal (PrCR) by acting as an "eat-me" signal for macrophages, directing their recognition and engulfment of dying, diseased, or unwanted cells. Recent findings have demonstrated that macrophages can transfer their own CALR onto exposed asialoglycans on target cells, marking them for PrCR. Despite the critical role CALR plays in this process, the molecular mechanisms behind its secretion by macrophages and the formation of binding sites on target cells remain unclear. Our findings show that CALR undergoes C-terminal cleavage upon secretion, producing a truncated form that functions as the active eat-me signal detectable on target cells. We identify cathepsins as potential proteases involved in this cleavage process. Furthermore, we demonstrate that macrophages release neuraminidases, which modify the surface of target cells and facilitate CALR binding. These insights reveal a coordinated mechanism through which lipopolysaccharide (LPS)-activated macrophages regulate CALR cleavage and neuraminidase activity to mark target cells for PrCR. How they recognize the cells to be targeted remains unknown.

}, keywords = {Animals, Apoptosis, Calreticulin, Humans, Lipopolysaccharides, Macrophages, Mice, Neuraminidase}, issn = {1091-6490}, doi = {10.1073/pnas.2426644122}, url = {https://doi.org/10.1073/pnas.2426644122}, author = {Banuelos, Allison and Baez, Michelle and Zhang, Allison and Y{\i}lmaz, Leyla and Kasberg, William and Volk, Regan and Georgeos, Nardin and Koren-Sedova, Elle and Le, Uyen and Burden, Andrew T and Marjon, Kristopher D and Lippincott-Schwartz, Jennifer and Zaro, Balyn W and Weissman, Irving L} } @article {69862, title = {Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Apr 17}, pages = {3401}, abstract = {

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

Preprint:\ 10.1101/2025.02.22.639343

}, keywords = {Animals, CRISPR-Cas Systems, Cryoelectron Microscopy, Endoplasmic Reticulum, HEK293 Cells, Hela Cells, Humans, Membrane Proteins, Mitochondria, Mitochondrial Membranes, Proteomics, Tacrolimus Binding Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-025-58538-3}, url = {https://doi.org/10.1038/s41467-025-58538-3}, author = {Nakamura, Koki and Aoyama-Ishiwatari, Saeko and Nagao, Takahiro and Paaran, Mohammadreza and Obara, Christopher J and Sakurai-Saito, Yui and Johnston, Jake and Du, Yudan and Suga, Shogo and Tsuboi, Masafumi and Nakakido, Makoto and Tsumoto, Kouhei and Kishi, Yusuke and Gotoh, Yukiko and Kwak, Chulhwan and Rhee, Hyun-Woo and Seo, Jeong Kon and Kosako, Hidetaka and Potter, Clint and Carragher, Bridget and Lippincott-Schwartz, Jennifer and Polleux, Franck and Hirabayashi, Yusuke} } @article {70194, title = {Pericyte and Endothelial Primary Cilia and Centrioles have Disparate Organization Across the Brain Microvasculature}, journal = {bioRxiv}, year = {2025}, month = {2025 Nov 19}, abstract = {

The brain microvascular functions are strongly influenced by the local microenvironment and cellular organization. Intracellular organelles, including primary cilia and centrioles, play critical roles in sensing and transmitting environmental cues and maintaining vascular integrity. However, their distribution across the brain vasculature remains poorly understood. In this study, we utilized publicly available large-volume electron microscopy datasets encompassing the cerebral vasculature from pial arterioles through parenchymal capillaries to pial venules. We systematically analyzed the cellular organization and characterized the distribution of primary cilia and centrioles in the mouse and human brain microvasculature. We found primary cilia exclusively on human cortical endothelial cells (ECs), indicating inter-species differences between mouse and human. Primary cilia were frequently present on mural cells (MCs, smooth muscle cells or pericytes) surrounding venules and capillaries but rarely observed on arterioles in both mouse and human brains. These MC primary cilia exhibited heterogeneity in ciliogenesis, including cells with ciliary pockets, surface cilia, and a hybrid configuration we refer as a partial pocket. In the mouse brain, many MC primary cilia were closely ensheathed by astrocytic endfeet and occasionally extended between them to establish proximity to synapses, whereas all primary cilia in the human brain were confined within the basal lamina. Our analysis of cellular density revealed similar EC densities between arterioles and venules in mice, but not in human. EC centrioles were consistently positioned against the direction of blood flow relative to the nuclei, suggesting that they may serve as a structural marker for flow direction. Collectively, these findings provide a comprehensive characterization of primary cilia and centrioles, highlighting distinct interspecies differences between mouse and human brain microvasculature. The proximity to neural cells and gradient distribution of these subcellular structures suggest that they may act as antennae for sensing mechanical and chemical signals within the brain microvascular environment.

}, doi = {10.1101/2025.11.19.689283}, url = {https://www.biorxiv.org/content/early/2025/11/19/2025.11.19.689283}, author = {Grubb, Soren and Chanddha, Vanshika and Lippincott-Schwartz, Jennifer and Ott, Carolyn and Mughal, Amreen} } @article {69756, title = {Periodic ER-plasma membrane junctions support long-range Ca signal integration in dendrites.}, journal = {Cell}, volume = {188}, year = {2025}, month = {01/2025}, pages = {484-500.e22}, abstract = {

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~{}1\ μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20\ μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

}, keywords = {Animals, Calcium, Calcium Signaling, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cell Membrane, Dendrites, Endoplasmic Reticulum, Hippocampus, Humans, Male, Mice, Rats, Ryanodine Receptor Calcium Release Channel}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.11.029}, author = {Benedetti, Lorena and Fan, Ruolin and Weigel, Aubrey V and Moore, Andrew S and Houlihan, Patrick R and Kittisopikul, Mark and Park, Grace and Petruncio, Alyson and Hubbard, Philip M and Pang, Song and Xu, C Shan and Hess, Harald F and Saalfeld, Stephan and Rangaraju, Vidhya and Clapham, David E and De Camilli, Pietro and Ryan, Timothy A and Lippincott-Schwartz, Jennifer} } @article {70082, title = {A protein tunnel helps stressed lysosomes swell.}, journal = {Science}, volume = {389}, year = {2025}, month = {2025 Aug 21}, pages = {782-783}, abstract = {

The endoplasmic reticulum donates lipids through a tunnel-like protein to help lysosomes expand.

}, keywords = {Adaptor Proteins, Signal Transducing, Animals, Endoplasmic Reticulum, Humans, Lipid Metabolism, Lysosomes}, issn = {1095-9203}, doi = {10.1126/science.aea5377}, url = {https://doi.org/10.1126/science.aea5377}, author = {Lippincott-Schwartz, Jennifer} } @article {70080, title = {RABGAP1 is a sensor that facilitates the sorting and processing of amyloid precursor protein}, journal = {The EMBO Journal}, year = {2025}, month = {2026 Aug 26}, pages = {1-20}, abstract = {

A hallmark of Alzheimer{\textquoteright}s disease (AD) is the accumulation of extracellular amyloid-β plaques in the brain. Amyloid-β is a 40{\textendash}42 amino acid peptide generated by proteolytic processing of amyloid precursor protein (APP) via membrane-bound proteases. APP is a transmembrane protein, and its trafficking to sites of proteolysis represents a rate-limiting step in AD progression. Although APP processing has been well-studied, its trafficking itinerary and machinery remain incompletely understood. To address this, we performed an unbiased interaction screen for interactors of the APP cytosolic tail. We identified previously characterised APP binders as well as novel interactors, including RABGAP1. We demonstrated that RABGAP1 partially co-localises with APP and directly interacts with a YENPTY motif in the APP cytosolic tail. Depletion or overexpression of RABGAP1 caused mistrafficking and misprocessing of endogenous APP in human and rodent neurons. This effect is dependent on the GAP activity of RABGAP1, demonstrating that RABGAP1 affects the trafficking of APP by modulating RAB activity on endosomal subdomains. This novel trafficking mechanism has implications for other NPXY cargoes and presents a possible therapeutic avenue to explore.

}, keywords = {Alzheimer{\textquoteright}s Disease, APP, RABGAP1, Trafficking}, doi = {10.1038/s44318-025-00530-0}, url = {https://www.embopress.org/doi/abs/10.1038/s44318-025-00530-0}, author = {Eden, Jessica and Kaufman, Jonathan G G and Pereira, Concei{\c c}{\~a}o and Fox, Eleanor and Cattin-Ortol{\'a}, Jerome and Benedetti, Lorena and Nieuwenhuis, Bart and Owen, David J and Lippincott-Schwartz, Jennifer and Munro, Sean and Gershlick, David C} } @article {70135, title = {Towards a unified framework for the function of endoplasmic reticulum exit sites.}, journal = {Nat Rev Mol Cell Biol}, year = {2025}, month = {2025 Sep 29}, abstract = {

Endoplasmic reticulum exit sites (ERES) are specialized, ribosome-free ER subdomains that serve as dynamic portals for COPII-mediated export of proteins from the ER. Beyond their role in the secretory pathway, ERES are implicated in diverse processes, including autophagy and the maturation of lipid droplets, highlighting their functional plasticity. ERES integrate cargo load, membrane tension and spatial cues to remodel their architecture and function in real time. This Roadmap synthesizes our current knowledge on the biogenesis, structural diversity and regulatory logic of ERES. We highlight key unanswered questions in the field, particularly concerning how ERES integrate signals to coordinate protein trafficking under varying cellular states. Finally, we propose a multidisciplinary framework - leveraging advances in high-resolution imaging, synthetic reconstitution and computational modelling - to delineate the principles governing the function and plasticity of ERES. Understanding these mechanisms holds significant potential for developing targeted therapeutic strategies in diseases linked to trafficking dysfunction.

}, issn = {1471-0080}, doi = {10.1038/s41580-025-00899-0}, url = {https://doi.org/10.1038/s41580-025-00899-0}, author = {Farhan, Hesso and Raote, Ishier and Campelo, Felix and Ge, Liang and Hirschberg, Koret and Forrester, Alison and Zanetti, Giulia and Lippincott-Schwartz, Jennifer and Pastor-Pareja, Jos{\'e} Carlos and Perez, Franck and Saito, Kota and Malhotra, Vivek} } @article {69812, title = {Vimentin filament transport and organization revealed by single-particle tracking and 3D FIB-SEM}, journal = {Journal of Cell Biology}, volume = {224}, year = {2025}, month = {2025 Apr 03}, pages = {e202406054}, abstract = {

Vimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant \~{}8\% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

bioRxiv Preprint:\ https://doi.org/10.1101/2024.06.10.598346

}, issn = {0021-9525}, doi = {10.1083/jcb.202406054}, url = {https://doi.org/10.1083/jcb.202406054}, author = {Renganathan, Bhuvanasundar and Moore, Andrew S. and Yeo, Wei-Hong and Petruncio, Alyson and Ackerman, David and Weigel, Aubrey V. and Team, The CellMap and Pasolli, H. Amalia and Xu, C. Shan and Shtengel, Gleb and Hess, Harald F. and Serpinskaya, Anna S. and Zhang, Hao F. and Lippincott-Schwartz, Jennifer and Gelfand, Vladimir I.} } @article {70049, title = {Vimentin intermediate filaments as structural and mechanical coordinators of mesenchymal cells.}, journal = {Nat Cell Biol}, year = {2025}, month = {2025 Aug 05}, abstract = {

Vimentin is a cytoskeletal intermediate filament protein that governs the form and function of mesenchymal cells, although the mechanistic details have been poorly understood. Here we highlight recent findings that reveal the diverse role of vimentin in dynamically organizing intracellular architecture and enhancing mechanical resilience. The exceptional deformability of vimentin can now be understood from its high-resolution three-dimensional structure resolved using cryo-electron microscopy. Vimentin also organizes the motion and positioning of numerous organelles, including mitochondria and the nucleus. Furthermore, it synergizes with the actin cytoskeleton to protect cells from extreme mechanical deformations. Finally, vimentin expression in epithelial-mesenchymal transitions has a functional role in tumour invasion analogous to embryonic development and wound healing. These recent developments emphasize the importance of understanding the multifaceted roles of vimentin intermediate filaments in human health and disease.

}, issn = {1476-4679}, doi = {10.1038/s41556-025-01713-x}, url = {https://doi.org/10.1038/s41556-025-01713-x}, author = {Guo, Ming and Wong, Ian Y and Moore, Andrew S and Medalia, Ohad and Lippincott-Schwartz, Jennifer and Weitz, David A and Goldman, Robert D} } @article {69351, title = {COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.}, journal = {Dev Cell}, year = {2024}, month = {2024 Apr 06}, abstract = {

Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca\ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed\ by\ super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism\ was ESCRT dependent and required\ ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In\ vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.

}, issn = {1878-1551}, doi = {10.1016/j.devcel.2024.03.027}, author = {Liao, Ya-Cheng and Pang, Song and Li, Wei-Ping and Shtengel, Gleb and Choi, Heejun and Schaefer, Kathy and Xu, C Shan and Lippincott-Schwartz, Jennifer} } @article {69224, title = {CSPP1 stabilizes microtubules by capping both plus and minus ends.}, journal = {Journal of Molecular Cell Biology}, year = {2024}, month = {2024 Feb 22}, abstract = {

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

}, issn = {1759-4685}, doi = {10.1093/jmcb/mjae007}, author = {Wang, Zhikai and Wang, Wenwen and Liu, Shuaiyu and Yang, Fengrui and Liu, Xu and Hua, Shasha and Zhu, Lijuan and Xu, Aoqing and Hill, Donald L and Wang, Dongmei and Jiang, Kai and Lippincott-Schwartz, Jennifer and Liu, Xing and Yao, Xuebiao} } @article {69223, title = {Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.}, journal = {Molecular Biology of the Cell}, year = {2024}, month = {2024 Feb 21}, pages = {mbcE23090379}, abstract = {

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based F{\"o}rster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

}, issn = {1939-4586}, doi = {10.1091/mbc.E23-09-0379}, author = {Dou, Zhen and Liu, Ran and Gui, Ping and Fu, Chuanhai and Lippincott-Schwartz, Jennifer and Yao, Xuebiao and Liu, Xing} } @article {69169, title = {Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.}, journal = {Nature}, volume = {626}, year = {2024}, month = {2024 Jan 24}, pages = {169-176}, abstract = {

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

}, keywords = {Amyotrophic Lateral Sclerosis, Endoplasmic Reticulum, Humans, Mitochondria, Mitochondrial Membranes, Signal Transduction, Vesicular Transport Proteins}, issn = {1476-4687}, doi = {10.1038/s41586-023-06956-y}, author = {Obara, Christopher J and Nixon-Abell, Jonathon and Moore, Andrew S and Riccio, Federica and Hoffman, David P and Shtengel, Gleb and Xu, C Shan and Schaefer, Kathy and Pasolli, H Amalia and Masson, Jean-Baptiste and Hess, Harald F and Calderon, Christopher P and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {69707, title = {Pearling Drives Mitochondrial DNA Nucleoid Distribution}, journal = {bioRxiv}, year = {2024}, month = {12/2024}, abstract = {

The regular distribution of mitochondrial DNA-containing nucleoids is essential for mitochondrial function and genome inheritance; however, the underlying mechanisms remain unknown. Our data reveal that mitochondria frequently undergo spontaneous and reversible pearling - a biophysical instability in which tubules undulate into regularly spaced beads. We discovered that pearling imposes a characteristic length scale, simultaneously mediating nucleoid disaggregation and establishing inter-nucleoid distancing with near-maximally achievable precision. Cristae invaginations play a dual role: lamellar cristae density determines pearling frequency and duration, and preserves the resulting nucleoid spacing after recovery. The distribution of mitochondrial genomes is thus fundamentally governed by the interplay between spontaneous pearling and cristae ultrastructure.

}, doi = {10.1101/2024.12.21.629917}, url = {https://www.biorxiv.org/content/early/2024/12/22/2024.12.21.629917}, author = {Landoni, Juan C. and Lycas, Matthew D. and Macuada, Josefa and Jaccard, Romeo and Obara, Christopher J. and Moore, Andrew S. and Ben Nejma, Sheda and Hoffman, David and Lippincott-Schwartz, Jennifer and Marshall, Wallace and Sturm, Gabriel and Manley, Suliana} } @article {69704, title = {Periodic ER-plasma membrane junctions support long-range Ca signal integration in dendrites.}, journal = {Cell}, year = {2024}, month = {2024 Dec 18}, abstract = {

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~{}1\ μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20\ μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.11.029}, author = {Benedetti, Lorena and Fan, Ruolin and Weigel, Aubrey V and Moore, Andrew S and Houlihan, Patrick R and Kittisopikul, Mark and Park, Grace and Petruncio, Alyson and Hubbard, Philip M and Pang, Song and Xu, C Shan and Hess, Harald F and Saalfeld, Stephan and Rangaraju, Vidhya and Clapham, David E and De Camilli, Pietro and Ryan, Timothy A and Lippincott-Schwartz, Jennifer} } @article {69705, title = {Permanent cilia loss during cerebellar granule cell neurogenesis involves withdrawal of cilia maintenance and centriole capping.}, journal = {Proc Natl Acad Sci U S A}, volume = {121}, year = {2024}, month = {2024 Dec 24}, pages = {e2408083121}, abstract = {

Brain neurons utilize the primary cilium as a privileged compartment to detect and respond to extracellular ligands such as Sonic hedgehog (SHH). However, cilia in cerebellar granule cell (GC) neurons disassemble during differentiation through ultrastructurally unique intermediates, a process we refer to as cilia deconstruction. In addition, mature neurons do not reciliate despite having docked centrioles. Here, we identify molecular changes that accompany cilia deconstruction and centriole docking in GC neurons. We used single cell transcriptomic and immunocytological analyses to compare the transcript levels and subcellular localization of proteins between progenitor, differentiating, and mature GCs. Differentiating GCs lacked transcripts for key activators of premitotic cilia resorption, indicating that cilia disassembly in differentiating cells is distinct from premitotic cilia resorption. Instead, during differentiation, transcripts of many genes required for cilia maintenance-specifically those encoding components of intraflagellar transport, pericentrosomal material, and centriolar satellites-decreased. The abundance of several corresponding proteins in and around cilia and centrosomes also decreased. These changes coincided with downregulation of SHH signaling prior to differentiation, even in a mutant with excessive SHH activation. Finally, mother centrioles in maturing granule neurons recruited the cap complex protein, CEP97. These data suggest that a global, developmentally programmed decrease in cilium maintenance in differentiating GCs mediates cilia deconstruction, while capping of docked mother centrioles prevents cilia regrowth and dysregulated SHH signaling. Our study provides mechanistic insights expanding our understanding of permanent cilia loss in multiple tissue-specific contexts.

}, keywords = {Animals, Cell Differentiation, Centrioles, Cerebellum, Cilia, Hedgehog Proteins, Mice, Neurogenesis, Neurons, Signal Transduction}, issn = {1091-6490}, doi = {10.1073/pnas.2408083121}, author = {Constable, Sandii and Ott, Carolyn M and Lemire, Andrew L and White, Kevin and Xun, Yu and Lim, Amin and Lippincott-Schwartz, Jennifer and Mukhopadhyay, Saikat} } @article {69136, title = {Permanent deconstruction of intracellular primary cilia in differentiating granule cell neurons.}, journal = {J Cell Biol.}, volume = {223}, year = {2024}, month = {2024 Aug 13}, pages = {e202404038}, abstract = {

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues.

}, doi = {10.1016/j.cub.2024.07.036}, url = {https://doi.org/10.1016/j.cub.2024.07.036 }, author = {Ott, Carolyn M and Constable, Sandii and Nguyen, Tri M and White, Kevin and Lee, Wei-Chung Allen and Lippincott-Schwartz, Jennifer and Mukhopadhyay, Saikat} } @article {69472, title = {The physical and cellular mechanism of structural color change in zebrafish.}, journal = {Proc Natl Acad Sci U S A}, volume = {121}, year = {2024}, month = {2024 Jun 04}, pages = {e2308531121}, abstract = {

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20{\textdegree} tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

}, keywords = {Animals, Color, Microscopy, Electron, Scanning, Norepinephrine, Pigmentation, Zebrafish, Zebrafish Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.2308531121}, author = {Gur, Dvir and Moore, Andrew S and Deis, Rachael and Song, Pang and Wu, Xufeng and Pinkas, Iddo and Deo, Claire and Iyer, Nirmala and Hess, Harald F and Hammer, John A and Lippincott-Schwartz, Jennifer} } @article {69687, title = {Structural dynamics of human ribosomes in situ reconstructed by exhaustive high-resolution template matching.}, journal = {Mol Cell}, year = {2024}, month = {2024 Nov 25}, abstract = {

Protein synthesis is central to life and requires the ribosome, which catalyzes the stepwise addition of amino acids to a polypeptide chain by undergoing a sequence of structural transformations. Here, we employed high-resolution template matching (HRTM) on cryoelectron microscopy (cryo-EM) images of directly cryofixed living cells to obtain a set of ribosomal configurations covering the entire elongation cycle, with each configuration occurring at its native abundance. HRTM{\textquoteright}s position and orientation precision and ability to detect small targets (\~{}300\ kDa) made it possible to order these configurations along the reaction coordinate and to reconstruct molecular features of any configuration along the elongation cycle. Visualizing the cycle{\textquoteright}s structural dynamics by combining a sequence of \>40 reconstructions into a 3D movie readily revealed component and ligand movements, some of them surprising, such as spring-like intramolecular motion, providing clues about the molecular mechanisms involved in some still mysterious steps during chain elongation.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2024.11.003}, author = {Rickgauer, J Peter and Choi, Heejun and Moore, Andrew S and Denk, Winfried and Lippincott-Schwartz, Jennifer} } @article {69491, title = {Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM}, journal = {bioRxiv}, year = {2024}, month = {2024 Jun 10}, abstract = {

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

}, doi = {10.1101/2024.06.10.598346}, url = {https://www.biorxiv.org/content/early/2024/06/10/2024.06.10.598346}, author = {Renganathan, Bhuvanasundar and Moore, Andrew S. and Yeo, Wei-Hong and Petruncio, Alyson and Ackerman, David and Wiegel, Aubrey and CellMap Team and Pasolli, H. Amalia and Xu, C. Shan and Hess, Harald F and Serpinskaya, Anna S. and Zhang, Hao F. and Lippincott-Schwartz, Jennifer and Gelfand, Vladimir I.} } @article {69459, title = {Ultrastructural differences impact cilia shape and external exposure across cell classes in the visual cortex}, journal = {Curr Biol}, year = {2024}, month = {2024 May 13}, abstract = {

A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2024.04.043}, author = {Ott, Carolyn M and Torres, Russel and Kuan, Tung-Sheng and Kuan, Aaron and Buchanan, JoAnn and Elabbady, Leila and Seshamani, Sharmishtaa and Bodor, Agnes L and Collman, Forrest and Bock, Davi D and Lee, Wei Chung and da Costa, Nuno Ma{\c c}arico and Lippincott-Schwartz, Jennifer} } @article {68935, title = {Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.}, journal = {bioRxiv}, year = {2023}, month = {2023 Aug 03}, abstract = {

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

}, doi = {10.1101/2023.08.01.551382}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J and Grimm, Jonathan and Lavis, Luke D and Singer, Robert H and Lippincott-Schwartz, Jennifer} } @article {68631, title = {Phase separation of Hippo signalling complexes.}, journal = {EMBO Journal}, volume = {42}, year = {2023}, month = {2023 Feb 20}, pages = {e112863}, abstract = {

The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and\ sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in\ vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.

}, issn = {1460-2075}, doi = {10.15252/embj.2022112863}, author = {Bonello, Teresa T and Cai, Danfeng and Fletcher, Georgina C and Wiengartner, Kyler and Pengilly, Victoria and Lange, Kimberly S and Liu, Zhe and Lippincott-Schwartz, Jennifer and Kavran, Jennifer M and Thompson, Barry J} } @article {68385, title = {Rationalized deep learning super-resolution microscopy for sustained live imaging of rapid subcellular processes.}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {2023 Mar 01}, pages = {367-77}, abstract = {

The goal when imaging bioprocesses with optical microscopy is to acquire the most spatiotemporal information with the least invasiveness. Deep neural networks have substantially improved optical microscopy, including image super-resolution and restoration, but still have substantial potential for artifacts. In this study, we developed rationalized deep learning (rDL) for structured illumination microscopy and lattice light sheet microscopy (LLSM) by incorporating prior knowledge of illumination patterns and, thereby, rationally guiding the network to denoise raw images. Here we demonstrate that rDL structured illumination microscopy eliminates spectral bias-induced resolution degradation and reduces model uncertainty by five-fold, improving the super-resolution information by more than ten-fold over other computational approaches. Moreover, rDL applied to LLSM enables self-supervised training by using the spatial or temporal continuity of noisy data itself, yielding results similar to those of supervised methods. We demonstrate the utility of rDL by imaging the rapid kinetics of motile cilia, nucleolar protein condensation during light-sensitive mitosis and long-term interactions between membranous and membrane-less organelles.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01471-3}, author = {Qiao, Chang and Li, Di and Liu, Yong and Zhang, Siwei and Liu, Kan and Liu, Chong and Guo, Yuting and Jiang, Tao and Fang, Chuyu and Li, Nan and Zeng, Yunmin and He, Kangmin and Zhu, Xueliang and Lippincott-Schwartz, Jennifer and Dai, Qionghai and Li, Dong} } @article {68798, title = {Structural Diversity within the Endoplasmic Reticulum-From the Microscale to the Nanoscale.}, journal = {Cold Spring Harbor Perspectives in Biology}, volume = {15}, year = {2023}, month = {2023 Jun 01}, abstract = {

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

}, keywords = {Animals, Endoplasmic Reticulum, Mammals, Microscopy, Electron, Nuclear Envelope}, issn = {1943-0264}, doi = {10.1101/cshperspect.a041259}, author = {Obara, Christopher J and Moore, Andrew S and Lippincott-Schwartz, Jennifer} } @article {68958, title = {Towards Generalizable Organelle Segmentation in Volume Electron Microscopy.}, journal = {Microscopy and Microanalysis}, volume = {29}, year = {2023}, month = {2023 Jul 22}, pages = {975}, keywords = {Organelles, Volume Electron Microscopy}, issn = {1435-8115}, doi = {10.1093/micmic/ozad067.487}, author = {Heinrich, Larissa and Patton, Will and Bennett, Davis and Ackerman, David and Park, Grace and Bogovic, John A and Eckstein, Nils and Petruncio, Alyson and Clements, Jody and Pang, Song and Shan Xu, C and Funke, Jan and Korff, Wyatt and Hess, Harald and Lippincott-Schwartz, Jennifer and Saalfeld, Stephan and Weigel, Aubrey} } @article {67844, title = {ER proteins decipher the tubulin code to regulate organelle distribution.}, journal = {Nature}, volume = {601}, year = {2022}, month = {2022 Jan 01}, pages = {132-138}, abstract = {

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum\ (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm, forming abundant contacts with other organelles. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-04204-9}, author = {Zheng, Pengli and Obara, Christopher J and Szczesna, Ewa and Nixon-Abell, Jonathon and Mahalingan, Kishore K and Roll-Mecak, Antonina and Lippincott-Schwartz, Jennifer and Blackstone, Craig} } @article {68512, title = {Oligodendrocyte precursor cells ingest axons in the mouse neocortex.}, journal = {Proceedings of the National Academies of Science of the U.S.A.}, volume = {119}, year = {2022}, month = {2022 Nov 29}, pages = {e2202580119}, abstract = {

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission\ electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

}, keywords = {Animals, Axons, Mice, Neocortex, Neurons, Oligodendrocyte Precursor Cells, Oligodendroglia}, issn = {1091-6490}, doi = {10.1073/pnas.2202580119}, author = {Buchanan, JoAnn and Elabbady, Leila and Collman, Forrest and Jorstad, Nikolas L and Bakken, Trygve E and Ott, Carolyn and Glatzer, Jenna and Bleckert, Adam A and Bodor, Agnes L and Brittain, Derrick and Bumbarger, Daniel J and Mahalingam, Gayathri and Seshamani, Sharmishtaa and Schneider-Mizell, Casey and Takeno, Marc M and Torres, Russel and Yin, Wenjing and Hodge, Rebecca D and Castro, Manuel and Dorkenwald, Sven and Ih, Dodam and Jordan, Chris S and Kemnitz, Nico and Lee, Kisuk and Lu, Ran and Macrina, Thomas and Mu, Shang and Popovych, Sergiy and Silversmith, William M and Tartavull, Ignacio and Turner, Nicholas L and Wilson, Alyssa M and Wong, William and Wu, Jingpeng and Zlateski, Aleksandar and Zung, Jonathan and Lippincott-Schwartz, Jennifer and Lein, Ed S and Seung, H Sebastian and Bergles, Dwight E and Reid, R Clay and da Costa, Nuno Ma{\c c}arico} } @article {68300, title = {Organization of translating secretome mRNAS on endoplasmic reticulum}, journal = {Biophysical Journal}, volume = {121}, year = {2022}, month = {2022 Feb 01}, pages = {33a}, abstract = {

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

}, issn = {00063495}, doi = {10.1016/j.bpj.2021.11.2550}, url = {https://linkinghub.elsevier.com/retrieve/pii/S0006349521035256}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J. and Grimm, Jonathan and Lavis, Luke D. and Singer, Robert H. and Lippincott-Schwartz, Jennifer} } @article {68369, title = {Structural Diversity within the Endoplasmic Reticulum-From the Microscale to the Nanoscale.}, journal = {Cold Spring Harbor Perspectives in Biology}, year = {2022}, month = {2022 Sep 19}, abstract = {

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

}, issn = {1943-0264}, doi = {10.1101/cshperspect.a041259}, author = {Obara, Christopher J and Moore, Andrew S and Lippincott-Schwartz, Jennifer} } @article {68167, title = {Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment.}, journal = {Cells}, volume = {11}, year = {2022}, month = {2022 Jun 12}, abstract = {

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.

}, keywords = {Apoptosis, Cluster Analysis, Fas Ligand Protein, fas Receptor, Fas-Associated Death Domain Protein, Receptors, Tumor Necrosis Factor}, issn = {2073-4409}, doi = {10.3390/cells11121908}, author = {Frazzette, Nicholas and Cruz, Anthony C and Wu, Xufeng and Hammer, John A and Lippincott-Schwartz, Jennifer and Siegel, Richard M and Sengupta, Prabuddha} } @article {68145, title = {Targeting LIPA independent of its lipase activity is a therapeutic strategy in solid tumors via induction of endoplasmic reticulum stress.}, journal = {Nature Cancer}, volume = {3}, year = {2022}, month = {2022 Jun 02}, pages = {866-884}, abstract = {

Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.

}, issn = {2662-1347}, doi = {10.1038/s43018-022-00389-8}, author = {Liu, Xihui and Viswanadhapalli, Suryavathi and Kumar, Shourya and Lee, Tae-Kyung and Moore, Andrew and Ma, Shihong and Chen, Liping and Hsieh, Michael and Li, Mengxing and Sareddy, Gangadhara R and Parra, Karla and Blatt, Eliot B and Reese, Tanner C and Zhao, Yuting and Chang, Annabel and Yan, Hui and Xu, Zhenming and Pratap, Uday P and Liu, Zexuan and Roggero, Carlos M and Tan, Zhenqiu and Weintraub, Susan T and Peng, Yan and Tekmal, Rajeshwar R and Arteaga, Carlos L and Lippincott-Schwartz, Jennifer and Vadlamudi, Ratna K and Ahn, Jung-Mo and Raj, Ganesh V} } @article {67177, title = {Actin cables and comet tails organize mitochondrial networks in mitosis.}, journal = {Nature}, volume = {591}, year = {2021}, month = {2021 Mar 03}, pages = {659-664}, abstract = {

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-03309-5}, author = {Moore, Andrew S and Coscia, Stephen M and Simpson, Cory L and Ortega, Fabian E and Wait, Eric C and Heddleston, John M and Nirschl, Jeffrey J and Obara, Christopher J and Guedes-Dias, Pedro and Boecker, C Alexander and Chew, Teng-Leong and Theriot, Julie A and Lippincott-Schwartz, Jennifer and Holzbaur, Erika L F} } @article {67304, title = {Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome}, journal = {bioRxiv}, year = {2021}, month = {05/2021}, abstract = {

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite{\textquoteright}s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2021.04.06.438745}, url = {https://doi.org/10.1101/2021.04.06.438745}, author = {Govind, Anitha P. and Jeyifous, Okunola and Russell, Theron A. and Yi, Zola and Weigel, Aubrey V. and Ramaprasad, Abhijit and Newell, Luke and Ramos, William and Valbuena, Fernando M. and Casler, Jason C. and Yan, Jing-Zhi and Glick, Benjamin S. and Swanson, Geoffrey T. and Lippincott-Schwartz, Jennifer and Green, William N.} } @article {67733, title = {Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome.}, journal = {eLife}, volume = {10}, year = {2021}, month = {2021 Sep 21}, abstract = {

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that -glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these -glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite{\textquoteright}s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.

}, issn = {2050-084X}, doi = {10.7554/eLife.68910}, author = {Govind, Anitha P and Jeyifous, Okunola and Russell, Theron A and Yi, Zola and Weigel, Aubrey V and Ramaprasad, Abhijit and Newell, Luke and Ramos, William and Valbuena, Fernando M and Casler, Jason C and Yan, Jing-Zhi and Glick, Benjamin S and Swanson, Geoffrey T and Lippincott-Schwartz, Jennifer and Green, William N} } @article {66893, title = {Biomolecular Condensates and Their Links to Cancer Progression.}, journal = {Trends in Biochemical Sciences}, year = {2021}, month = {2021 Feb 10}, abstract = {

Liquid-liquid phase separation (LLPS) has emerged in recent years as an important physicochemical process for organizing diverse processes within cells via the formation of membraneless organelles termed biomolecular condensates. Emerging evidence now suggests that the formation and regulation of biomolecular condensates are also intricately linked to cancer formation and progression. We review the most recent literature linking the existence and/or dissolution of biomolecular condensates to different hallmarks of cancer formation and progression. We then discuss the opportunities that this condensate perspective provides for cancer research and the development of novel therapeutic approaches, including the perturbation of condensates by small-molecule inhibitors.

}, issn = {0968-0004}, doi = {10.1016/j.tibs.2021.01.002}, author = {Cai, Danfeng and Liu, Zhe and Lippincott-Schwartz, Jennifer} } @article {67312, title = {Biomolecular Condensates and Their Links to Cancer Progression.}, journal = {Trends in Biochemical Sciences}, volume = {46}, year = {2021}, month = {2021 Jul 01}, pages = {535-549}, abstract = {

Liquid-liquid phase separation (LLPS) has emerged in recent years as an important physicochemical process for organizing diverse processes within cells via the formation of membraneless organelles termed biomolecular condensates. Emerging evidence now suggests that the formation and regulation of biomolecular condensates are also intricately linked to cancer formation and progression. We review the most recent literature linking the existence and/or dissolution of biomolecular condensates to different hallmarks of cancer formation and progression. We then discuss the opportunities that this condensate perspective provides for cancer research and the development of novel therapeutic approaches, including the perturbation of condensates by small-molecule inhibitors.

}, issn = {0968-0004}, doi = {10.1016/j.tibs.2021.01.002}, author = {Cai, Danfeng and Liu, Zhe and Lippincott-Schwartz, Jennifer} } @article {67233, title = {ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.}, journal = {Cell}, volume = {184}, year = {2021}, month = {2021 Apr 29}, pages = {2412}, abstract = {

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30\% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2021.03.035}, author = {Weigel, Aubrey V and Chang, Chi-Lun and Shtengel, Gleb and Xu, C Shan and Hoffman, David P and Freeman, Melanie and Iyer, Nirmala and Aaron, Jesse and Khuon, Satya and Bogovic, John and Qiu, Wei and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {67244, title = {A general method to improve fluorophores using deuterated auxochromes.}, journal = {JACS Au}, volume = {1}, year = {2021}, month = {2021 May 24}, pages = {690-6}, abstract = {

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways{\textemdash}thereby increasing the brightness and photostability of fluorophores{\textemdash}are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

}, issn = {2691-3704}, doi = {10.1021/jacsau.1c00006}, url = {https://doi.org/10.1021/jacsau.1c00006}, author = {Grimm, Jonathan B. and Xie, Liangqi and Casler, Jason C. and Patel, Ronak and Tkachuk, Ariana N. and Falco, Natalie and Choi, Heejun and Lippincott-Schwartz, Jennifer and Brown, Timothy A. and Glick, Benjamin S. and Liu, Zhe and Lavis, Luke D.} } @article {66002, title = {Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.}, journal = {Journal of Cell Biology}, volume = {220}, year = {2021}, month = {2021 Feb 01}, abstract = {

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

}, issn = {1540-8140}, doi = {10.1083/jcb.202006180}, author = {Kanfer, Gil and Sarraf, Shireen A and Maman, Yaakov and Baldwin, Heather and Dominguez-Martin, Eunice and Johnson, Kory R and Ward, Michael E and Kampmann, Martin and Lippincott-Schwartz, Jennifer and Youle, Richard J} } @article {67339, title = {Lipid droplets in the nervous system.}, journal = {Journal of Cell Biology}, volume = {220}, year = {2021}, month = {2021 Jul 05}, abstract = {

Lipid droplets are dynamic intracellular lipid storage organelles that respond to the physiological state of cells. In addition to controlling cell metabolism, they play a protective role for many cellular stressors, including oxidative stress. Despite prior descriptions of lipid droplets appearing in the brain as early as a century ago, only recently has the role of lipid droplets in cells found in the brain begun to be understood. Lipid droplet functions have now been described for cells of the nervous system in the context of development, aging, and an increasing number of neuropathologies. Here, we review the basic mechanisms of lipid droplet formation, turnover, and function and discuss how these mechanisms enable lipid droplets to function in different cell types of the nervous system under healthy and pathological conditions.

}, issn = {1540-8140}, doi = {10.1083/jcb.202102136}, author = {Ralhan, Isha and Chang, Chi-Lun and Lippincott-Schwartz, Jennifer and Ioannou, Maria S} } @article {67753, title = {An open-access volume electron microscopy atlas of whole cells and tissues.}, journal = {Nature}, volume = {599}, year = {2021}, month = {2021 Nov 1}, pages = {147-51}, abstract = {

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels. Owing to advances in the\ precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm\ voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and\ pose questions.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-03992-4}, author = {Xu, C Shan and Pang, Song and Shtengel, Gleb and M{\"u}ller, Andreas and Ritter, Alex T and Hoffman, Huxley K and Takemura, Shin-ya and Lu, Zhiyuan and Pasolli, H Amalia and Iyer, Nirmala and Chung, Jeeyun and Bennett, Davis and Weigel, Aubrey V and Freeman, Melanie and Van Engelenburg, Schuyler B and Walther, Tobias C and Farese, Robert V and Lippincott-Schwartz, Jennifer and Mellman, Ira and Solimena, Michele and Hess, Harald F} } @article {66024, title = {RNA transport and local translation in neurodevelopmental and neurodegenerative disease.}, journal = {Nature Neuroscience}, volume = {24}, year = {2021}, month = {2021 May 01}, pages = {622-32}, abstract = {

Neurons decentralize protein synthesis from the cell body to support the active metabolism of remote dendritic and axonal compartments. The neuronal RNA transport apparatus, composed of cis-acting RNA regulatory elements, neuronal transport granule proteins, and motor adaptor complexes, drives the long-distance RNA trafficking required for local protein synthesis. Over the past decade, advances in human genetics, subcellular biochemistry, and high-resolution imaging have implicated each member of the apparatus in several neurodegenerative diseases, establishing failed RNA transport and associated processes as a unifying pathomechanism. In this review, we deconstruct the RNA transport apparatus, exploring each constituent{\textquoteright}s role in RNA localization and illuminating their unique contributions to neurodegeneration.

}, issn = {1546-1726}, doi = {10.1038/s41593-020-00785-2}, author = {Fernandopulle, Michael S and Lippincott-Schwartz, Jennifer and Ward, Michael E} } @article {67750, title = {Whole-cell organelle segmentation in volume electron microscopy.}, journal = {Nature}, volume = {599}, year = {2021}, month = {2021 Nov 01}, pages = {141-46}, abstract = {

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM). We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, {\textquoteright}OpenOrganelle{\textquoteright}, to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-03977-3}, author = {Heinrich, Larissa and Bennett, Davis and Ackerman, David and Park, Woohyun and Bogovic, John and Eckstein, Nils and Petruncio, Alyson and Clements, Jody and Pang, Song and Xu, C Shan and Funke, Jan and Korff, Wyatt and Hess, Harald F and Lippincott-Schwartz, Jennifer and Saalfeld, Stephan and Weigel, Aubrey V} } @article {67646, title = {YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {2021 Jul 23}, pages = {4502}, abstract = {

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells.\ RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-24708-2}, author = {Feliciano, Daniel and Ott, Carolyn M and Isabel Espinosa Medina and Weigel, Aubrey V and Benedetti, Lorena and Milano, Kristin M and Tang, Zhonghua and Lee, Tzumin and Kliman, Harvey J and Guller, Seth M and Lippincott-Schwartz, Jennifer} } @article {65281, title = {Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.}, journal = {Science}, volume = {367}, year = {2020}, month = {2020 Jan 17}, abstract = {

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

}, issn = {1095-9203}, doi = {10.1126/science.aaz5357}, author = {Hoffman, David P and Shtengel, Gleb and Xu, C Shan and Campbell, Kirby R and Freeman, Melanie and Wang, Lei and Milkie, Daniel E and Pasolli, H Amalia and Iyer, Nirmala and Bogovic, John A and Stabley, Daniel R and Shirinifard, Abbas and Pang, Song and Peale, David and Schaefer, Kathy and Pomp, Wim and Chang, Chi-Lun and Lippincott-Schwartz, Jennifer and Kirchhausen, Tom and Solecki, David J and Betzig, Eric and Hess, Harald F} } @article {65367, title = {ER membranes exhibit phase behavior at sites of organelle contact.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, year = {2020}, month = {2020 March 31}, pages = {7225-7235}, abstract = {

The endoplasmic reticulum (ER) is the site of synthesis of secretory and membrane proteins and contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner. How the ER spatially segregates its numerous and diverse functions, including positioning nanoscopic contact sites with other organelles, is unclear. We demonstrate that hypotonic swelling of cells converts the ER and other membrane-bound organelles into micrometer-scale large intracellular vesicles (LICVs) that retain luminal protein content and maintain contact sites with each other through localized organelle tethers. Upon cooling, ER-derived LICVs phase-partition into microscopic domains having different lipid-ordering characteristics, which is reversible upon warming. Ordered ER lipid domains mark contact sites with ER and mitochondria, lipid droplets, endosomes, or plasma membrane, whereas disordered ER lipid domains mark contact sites with lysosomes or peroxisomes. Tethering proteins concentrate at ER{\textendash}organelle contact sites, allowing time-dependent behavior of lipids and proteins to be studied at these sites. These findings demonstrate that LICVs provide a useful model system for studying the phase behavior and interactive properties of organelles in intact cells.

}, issn = {0027-8424}, doi = {10.1073/pnas.1910854117}, url = {http://www.pnas.org/lookup/doi/10.1073/pnas.1910854117}, author = {King, Christopher and Sengupta, Prabuddha and Seo, Arnold Y. and Lippincott-Schwartz, Jennifer} } @article {65858, title = {The evolution of a cell biologist.}, journal = {Molecular Biology of the Cell}, volume = {31}, year = {2020}, month = {2020 Dec 01}, pages = {2763-2767}, abstract = {

I am honored and humbled to receive the E. B. Wilson Medal and happy to share some reflections on my journey as a cell biologist. It took me a while to realize that my interest in biology would center on how cells are spatially and dynamically organized. From an initial fascination with cellular structures I came to appreciate that cells exhibit dynamism across all scales-from their molecules, to molecular complexes, to organelles. Uncovering the principles of this dynamism, including new ways to observe and quantify it, has been the guiding star of my work.

}, issn = {1939-4586}, doi = {10.1091/mbc.E20-09-0603}, author = {Lippincott-Schwartz, Jennifer} } @article {65667, title = {A general method to optimize and functionalize red-shifted rhodamine dyes.}, journal = {Nature Methods}, year = {2020}, month = {2020 Jul 27}, abstract = {

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted {\textquoteright}Janelia Fluor{\textquoteright} (JF) dyes useful for biological imaging experiments in cells and in vivo.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0909-6}, author = {Grimm, Jonathan B and Tkachuk, Ariana N and Xie, Liangqi and Choi, Heejun and Mohar, Boaz and Falco, Natalie and Schaefer, Kathy and Patel, Ronak and Zheng, Qinsi and Liu, Zhe and Lippincott-Schwartz, Jennifer and Brown, Timothy A and Lavis, Luke D} } @article {65896, title = {In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning.}, journal = {Nature Communications}, volume = {11}, year = {2020}, month = {2020 Dec 15}, pages = {6391}, abstract = {

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish{\textquoteright}s color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

}, keywords = {Animals, Cell Differentiation, Cell Proliferation, Chromatophores, Microphthalmia-Associated Transcription Factor, Mutagenesis, Skin, Skin Pigmentation, Transcriptome, X-Ray Diffraction, Zebrafish, Zebrafish Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-020-20088-1}, author = {Gur, Dvir and Bain, Emily J and Johnson, Kory R and Aman, Andy J and Pasoili, H Amalia and Flynn, Jessica D and Allen, Michael C and Deheyn, Dimitri D and Lee, Jennifer C and Lippincott-Schwartz, Jennifer and Parichy, David M} } @article {65631, title = {Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking}, journal = {Matrix Biology}, volume = {93}, year = {2020}, month = {2020 Nov 01}, pages = {79-94}, abstract = {

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.

}, issn = {0945053X}, doi = {10.1016/j.matbio.2020.06.002}, url = {https://linkinghub.elsevier.com/retrieve/pii/S0945053X20300676}, author = {Omari, Shakib and Makareeva, Elena and Gorrell, Laura and Jarnik, Michal and Lippincott-Schwartz, Jennifer and Leikin, Sergey} } @article {65318, title = {Nicotine exposure and neuronal activity regulate Golgi membrane dispersal and distribution}, journal = {bioRxiv}, year = {2020}, month = {2020 Feb 26}, abstract = {

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of α4β2-type nicotinic acetylcholine receptors (α4β2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required α4β2R expression. Similar nicotine-induced changes occurred\ in vivo\ at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotine{\textquoteright}s addictive effects. Characterization\ in vitro\ demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger {\textquotedblleft}ministacks{\textquotedblright}, similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of α4β2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

}, author = {Govind, Anitha P. and Jeyifous, Okunola and Russell, Theron A. and Vaasjo, Lee O. and Yi, Zola and Weigel, Aubrey V. and Newell, Luke and Koranda, Jessica L. and Singh, Karanveer and Valbuena, Fernando and Glick, Benjamin S. and Mukherjee, Jogeshwar and Lippincott-Schwartz, Jennifer and Zhuang, Xiaoxi and Green, William N.} } @article {65654, title = {Revisiting Membrane Microdomains and Phase Separation: A Viral Perspective}, journal = {Viruses}, volume = {12}, year = {2020}, month = {2020 Jul 10}, pages = {745}, abstract = {

Retroviruses selectively incorporate a specific subset of host cell proteins and lipids into their outer membrane when they bud out from the host plasma membrane. This specialized viral membrane composition is critical for both viral survivability and infectivity. Here, we review recent findings from live cell imaging of single virus assembly demonstrating that proteins and lipids sort into the HIV retroviral membrane by a mechanism of lipid-based phase partitioning. The findings showed that multimerizing HIV Gag at the assembly site creates a liquid-ordered lipid phase enriched in cholesterol and sphingolipids. Proteins with affinity for this specialized lipid environment partition into it, resulting in the selective incorporation of proteins into the nascent viral membrane. Building on this and other work in the field, we propose a model describing how HIV Gag induces phase separation of the viral assembly site through a mechanism involving transbilayer coupling of lipid acyl chains and membrane curvature changes. Similar phase-partitioning pathways in response to multimerizing structural proteins likely help sort proteins into the membranes of other budding structures within cells.

}, doi = {10.3390/v12070745}, url = {https://www.mdpi.com/1999-4915/12/7/745}, author = {Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer} } @article {64918, title = {De novo design of tunable, pH-driven conformational changes.}, journal = {Science}, volume = {364}, year = {2019}, month = {2019 May 17}, pages = {658-64}, abstract = {

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.

}, doi = {10.1126/science.aav7897}, author = {Boyken, Scott E and Benhaim, Mark A and Busch, Florian and Jia, Mengxuan and Back, Matthew J and Choi, Heejun and Klima, Jason C and Chen, Zibo and Walkey, Carl and Mileant, Alexander and Sahasrabuddhe, Aniruddha and Wei, Kathy Y and Hodge, Edgar A and Byron, Sarah and Quijano-Rubio, Alfredo and Sankaran, Banumathi and King, Neil P and Lippincott-Schwartz, Jennifer and Wysocki, Vicki H and et al} } @article {64959, title = {Fate plasticity and reprogramming in genetically distinct populations of leucophores.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, year = {2019}, month = {2019 Jun 11}, pages = {11806-11}, abstract = {

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of fishes, including the zebrafish , we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

}, issn = {1091-6490}, doi = {10.1073/pnas.1901021116}, author = {Lewis, Victor M and Saunders, Lauren M and Larson, Tracy A and Bain, Emily J and Sturiale, Samantha L and Gur, Dvir and Chowdhury, Sarwat and Flynn, Jessica D and Allen, Michael C and Deheyn, Dimitri D and Lee, Jennifer C and Simon, Julian A and Lippincott-Schwartz, Jennifer and Raible, David W and Parichy, David M} } @article {64863, title = {A lipid-based partitioning mechanism for selective incorporation of proteins into membranes of HIV particles.}, journal = {Nature Cell Biology}, volume = {21}, year = {2019}, month = {2019 Apr}, pages = {452-461}, abstract = {

Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodeling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.

}, issn = {1476-4679}, doi = {10.1038/s41556-019-0300-y}, author = {Sengupta, Prabuddha and Seo, Arnold Y and Pasolli, H Amalia and Song, Yul Eum and Johnson, Marc and Lippincott-Schwartz, Jennifer} } @article {64987, title = {NDP52 tunes cortical actin interaction with astral microtubules for accurate spindle orientation.}, journal = {Cell Research}, volume = {29}, year = {2019}, month = {2019 Jun 14}, pages = {666-79}, abstract = {

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

}, issn = {1748-7838}, doi = {10.1038/s41422-019-0189-9}, author = {Yu, Huijuan and Yang, Fengrui and Dong, Peng and Liao, Shanhui and Liu, Wei R and Zhao, Gangyin and Qin, Bo and Dou, Zhen and Liu, Zhe and Liu, Wei and Zang, Jianye and Lippincott-Schwartz, Jennifer and Liu, Xing and Yao, Xuebiao} } @article {65067, title = {Neuron-astrocyte metabolic coupling protects against activity-induced fatty acid toxicity.}, journal = {Cell}, volume = {177}, year = {2019}, month = {2019 May 30}, pages = {1522-1535.e14}, abstract = {

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.04.001}, author = {Ioannou, Maria S and Jackson, Jesse and Sheu, Shu-Hsien and Chang, Chi-Lun and Weigel, Aubrey V and Liu, Hui and Pasolli, H Amalia and Xu, C Shan and Pang, Song and Matthies, Doreen and Hess, Harald F and Lippincott-Schwartz, Jennifer and Liu, Zhe} } @article {65139, title = {A neuron-glia Co-culture system for studying intercellular lipid transport.}, journal = {Curr Protoc Cell Biol}, volume = {84}, year = {2019}, month = {2019 Sep 01}, pages = {e95}, abstract = {

Neurons and glia operate in a highly coordinated fashion in the brain. Although glial cells have long been known to supply lipids to neurons via lipoprotein particles, new evidence reveals that lipid transport between neurons and glia is bidirectional. Here, we describe a co-culture system to study transfer of lipids and lipid-associated proteins from neurons to glia. The assay entails culturing neurons and glia on separate coverslips, pulsing the neurons with fluorescently labeled fatty acids, and then incubating the coverslips together. As astrocytes internalize and store neuron-derived fatty acids in lipid droplets, analyzing the number, size, and fluorescence intensity of lipid droplets containing the fluorescent fatty acids provides an easy and quantifiable measure of fatty acid transport. {\textcopyright} 2019 The Authors.

}, issn = {1934-2616}, doi = {10.1002/cpcb.95}, author = {Ioannou, Maria S and Liu, Zhe and Lippincott-Schwartz, Jennifer} } @article {65245, title = {Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression.}, journal = {Nature Cell Biology}, volume = {21}, year = {2019}, month = {2019 Dec}, pages = {1578-1589}, abstract = {

Yes-associated protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized the YAP transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced the transcription of YAP-specific proliferation genes. Super-resolution imaging using assay for transposase-accessible chromatin with photoactivated localization microscopy revealed that the YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA polymerase II, the accessible chromatin domains later acquired RNA polymerase II, transcribing RNA. The removal of the intrinsically-disordered YAP transcription activation domain prevented the formation of YAP condensates and diminished downstream YAP signalling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

}, issn = {1476-4679}, doi = {10.1038/s41556-019-0433-z}, author = {Cai, Danfeng and Feliciano, Daniel and Dong, Peng and Flores, Eduardo and Gruebele, Martin and Porat-Shliom, Natalie and Sukenik, Shahar and Liu, Zhe and Lippincott-Schwartz, Jennifer} } @article {65176, title = {Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.}, journal = {ACS Central Science}, volume = {5}, year = {2019}, month = {2019 Sep 25}, pages = {1602-1613}, abstract = {

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.9b00676}, author = {Zheng, Qinsi and Ayala, Anthony X and Chung, Inhee and Weigel, Aubrey V and Ranjan, Anand and Falco, Natalie and Grimm, Jonathan B and Tkachuk, Ariana N and Wu, Carl and Lippincott-Schwartz, Jennifer and Singer, Robert H and Lavis, Luke D} } @article {65025, title = {Regulation of plasma membrane nanodomains of the water channel aquaporin-3 revealed by fixed and live photoactivated localization microscopy.}, journal = {Nano Letters}, volume = {19}, year = {2019}, month = {2019 Feb 13}, pages = {699-707}, abstract = {

Several aquaporin (AQP) water channels are short-term regulated by the messenger cyclic adenosine monophosphate (cAMP), including AQP3. Bulk measurements show that cAMP can change diffusive properties of AQP3; however, it remains unknown how elevated cAMP affects AQP3 organization at the nanoscale. Here we analyzed AQP3 nano-organization following cAMP stimulation using photoactivated localization microscopy (PALM) of fixed cells combined with pair correlation analysis. Moreover, in live cells, we combined PALM acquisitions of single fluorophores with single-particle tracking (spt-PALM). These analyses revealed that AQP3 tends to cluster and that the diffusive mobility is confined to nanodomains with radii of \~{}150 nm. This domain size increases by \~{}30\% upon elevation of cAMP, which, however, is not accompanied by a significant increase in the confined diffusion coefficient. This regulation of AQP3 organization at the nanoscale may be important for understanding the mechanisms of water AQP3-mediated water transport across plasma membranes.

}, keywords = {Animals, Aquaporin 3, Cell Membrane, Cyclic AMP, Diffusion, Dogs, Epithelial Cells, Madin Darby Canine Kidney Cells, Microscopy, Fluorescence, Photochemical Processes}, issn = {1530-6992}, doi = {10.1021/acs.nanolett.8b03721}, author = {Arnspang, Eva C and Sengupta, Prabuddha and Mortensen, Kim I and Jensen, Helene H and Hahn, Ute and Jensen, Eva B V and Lippincott-Schwartz, Jennifer and Nejsum, Lene N} } @article {65156, title = {RNA granules hitchhike on lysosomes for long-distance transport, Using annexin A11 as a molecular tether.}, journal = {Cell}, volume = {179}, year = {2019}, month = {2019 Sep 19}, pages = {147-164.e20}, abstract = {

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In\ vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.08.050}, author = {Liao, Ya-Cheng and Fernandopulle, Michael S and Wang, Guozhen and Choi, Heejun and Hao, Ling and Drerup, Catherine M and Patel, Rajan and Qamar, Seema and Nixon-Abell, Jonathon and Shen, Yi and Meadows, William and Vendruscolo, Michele and Knowles, Tuomas P J and Nelson, Matthew and Czekalska, Magdalena A and Musteikyte, Greta and Gachechiladze, Mariam A and Stephens, Christina A and Pasolli, H Amalia and Forrest, Lucy R and St George-Hyslop, Peter and Lippincott-Schwartz, Jennifer and Ward, Michael E} } @article {64795, title = {Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.}, journal = {Journal of Cell Biology}, volume = {218}, year = {2019}, month = {2019 Jun 21}, pages = {2583-99}, abstract = {

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two inter-related mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1 and CHMP1B modifying LD membrane morphology. Furthermore, M1 Spastin, IST1 and CHMP1B are all required to relieve LDs of lipid peroxidation. The roles of M1 Spastin in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may help explain the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

}, doi = {10.1101/544023}, author = {Chang, Chi-Lun and Weigel, Aubrey V and Ioannou, Maria S and Pasolli, H Amalia and Xu, C Shan and Peale, David R and Shtengel, Gleb and Freeman, Melanie and Hess, Harald F and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {49419, title = {The development and enhancement of FRAP as a key tool for investigating protein dynamics.}, journal = {Biophysical Journal}, volume = {115}, year = {2018}, month = {2018 Aug 17}, pages = {1146-55}, abstract = {

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et\ al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP{\textquoteright}s reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2018.08.007}, author = {Lippincott-Schwartz, Jennifer and Snapp, Erik Lee and Phair, Robert D} } @article {49314, title = {Interacting organelles.}, journal = {Current Opinion in Cell Biology}, volume = {53}, year = {2018}, month = {2018 Aug}, pages = {84-91}, abstract = {

Eukaryotic cells are organized into membrane-bound organelles. These organelles communicate with one another through vesicular trafficking pathways and membrane contact sites (MCSs). MCSs are sites of close apposition between two or more organelles that play diverse roles in the exchange of metabolites, lipids and proteins. Organelle interactions at MCSs also are important for organelle division and biogenesis. For example, the division of several organelles, including mitochondria and endosomes, seem to be regulated by contacts with the endoplasmic reticulum (ER). Moreover, the biogenesis of autophagosomes and peroxisomes involves contributions from the ER and multiple other cellular compartments. Thus, organelle-organelle interactions allow cells to alter the shape and activities of their membrane-bound compartments, allowing them to cope with different developmental and environmental conditions.

}, doi = {10.1016/j.ceb.2018.06.003}, author = {Cohen, Sarah and Valm, Alex M and Lippincott-Schwartz, Jennifer} } @article {49291, title = {Monitoring the effects of pharmacological reagents on mitochondrial morphology.}, journal = {Current Protocols in Cell Biology}, volume = {79}, year = {2018}, month = {2018 Jun}, pages = {e45}, abstract = {

This protocol describes how to apply appropriate pharmacological controls to induce mitochondrial fusion or fission in studies of mitochondria morphology for four different mammalian cell types, HepG2 human liver hepatocellular carcinoma cells, MCF7 human breast adenocarcinoma cells, HEK293 human embryonic kidney cells, and collagen sandwich culture of primary rat hepatocytes. The protocol provides methods of treating cells with these pharmacological controls, staining mitochondria with commercially available MitoTracker Green and TMRE dyes, and imaging the mitochondrial morphology in live cells using a confocal fluorescent microscope. It also describes the cell culture methods needed for this protocol. {\textcopyright} 2018 by John Wiley \& Sons, Inc.

}, issn = {1934-2616}, doi = {10.1002/cpcb.45}, author = {Fu, Dong and Lippincott-Schwartz, Jennifer} } @article {49370, title = {mTOR-dependent phosphorylation controls TFEB nuclear export.}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {2018 Aug 17}, pages = {3312}, abstract = {

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data on TFEB nucleo-cytoplasmic shuttling suggest an unpredicted role of mTOR in nuclear export.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-05862-6}, author = {Napolitano, Gennaro and Esposito, Alessandra and Choi, Heejun and Matarese, Maria and Benedetti, Valerio and Di Malta, Chiara and Monfregola, Jlenia and Medina, Diego Luis and Lippincott-Schwartz, Jennifer and Ballabio, Andrea} } @article {49351, title = {Multispectral live-cell imaging.}, journal = {Current Protocols in Cell Biology}, volume = {79}, year = {2018}, month = {2018 Jun}, pages = {e46}, abstract = {

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. {\textcopyright} 2018 by John Wiley \& Sons, Inc.

}, issn = {1934-2616}, doi = {10.1002/cpcb.46}, author = {Cohen, Sarah and Valm, Alex M and Lippincott-Schwartz, Jennifer} } @article {49527, title = {MYC induces a hybrid energetics program early in cell reprogramming.}, journal = {Stem Cell Reports}, volume = {11}, year = {2018}, month = {2018 Dec 11}, pages = {1479-92}, abstract = {

Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2018.10.018}, author = {Prieto, Javier and Seo, Arnold Y and Le{\'o}n, Marian and Santacatterina, Fulvio and Torresano, Laura and Palomino-Sch{\"a}tzlein, Martina and Gim{\'e}nez, Karen and Vallet-S{\'a}nchez, Azahara and Ponsoda, Xavier and Pineda-Lucena, Antonio and Cuezva, Jos{\'e} M and Lippincott-Schwartz, Jennifer and Torres, Josema} } @article {49457, title = {Noncanonical autophagy at ER exit sites regulates procollagen turnover.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2018}, month = {2018 Oct 04}, pages = {E10099-108}, abstract = {

Type I collagen is the main component of bone matrix and other connective tissues. Rerouting of its procollagen precursor to a degradative pathway is crucial for osteoblast survival in pathologies involving excessive intracellular buildup of procollagen that is improperly folded and/or trafficked. What cellular mechanisms underlie this rerouting remains unclear. To study these mechanisms, we employed live-cell imaging and correlative light and electron microscopy (CLEM) to examine procollagen trafficking both in wild-type mouse osteoblasts and osteoblasts expressing a bone pathology-causing mutant procollagen. We found that although most procollagen molecules successfully trafficked through the secretory pathway in these cells, a subpopulation did not. The latter molecules appeared in numerous dispersed puncta colocalizing with COPII subunits, autophagy markers and ubiquitin machinery, with more puncta seen in mutant procollagen-expressing cells. Blocking endoplasmic reticulum exit site (ERES) formation suppressed the number of these puncta, suggesting they formed after procollagen entry into ERESs. The punctate structures containing procollagen, COPII, and autophagic markers did not move toward the Golgi but instead were relatively immobile. They appeared to be quickly engulfed by nearby lysosomes through a bafilomycin-insensitive pathway. CLEM and fluorescence recovery after photobleaching experiments suggested engulfment occurred through a noncanonical form of autophagy resembling microautophagy of ERESs. Overall, our findings reveal that a subset of procollagen molecules is directed toward lysosomal degradation through an autophagic pathway originating at ERESs, providing a mechanism to remove excess procollagen from cells.

}, issn = {1091-6490}, doi = {10.1073/pnas.1814552115}, author = {Omari, Shakib and Makareeva, Elena and Roberts-Pilgrim, Anna and Mirigian, Lynn and Jarnik, Michal and Ott, Carolyn and Lippincott-Schwartz, Jennifer and Leikin, Sergey} } @article {49354, title = {Triggered cell-cell fusion assay for cytoplasmic and organelle intermixing studies.}, journal = {Current Protocols in Cell Biology}, volume = {81}, year = {2018}, month = {2018 Aug 13}, pages = {e61}, abstract = {

Different multicellular organisms undergo cell-cell fusion to form functional syncytia that support specialized functions necessary for proper development and survival. For years, monitoring the structural consequences of this process using live-cell imaging has been challenging due to the unpredictable timing of cell fusion events in tissue systems. Here we present a triggered vesicular stomatitis virus G-protein (VSV-G)-mediated cell-cell fusion assay that can be used to synchronize fusion between cells. This allows the study of cellular changes that occur during cell fusion. The process is induced using a fast wash of low pH isotonic buffer, promoting the fusion of plasma membranes of two or more adjacent cells within seconds. This approach is suitable for studying mixing of small cytoplasmic molecules between fusing cells as well as changes in organelle distribution and dynamics. {\textcopyright} 2018 by John Wiley \& Sons, Inc.

}, issn = {1934-2616}, doi = {10.1002/cpcb.61}, author = {Feliciano, Daniel and Nixon-Abell, Jonathon and Lippincott-Schwartz, Jennifer} } @article {49512, title = {Visualizing intracellular organelle and cytoskeletal interactions at nanoscale resolution on millisecond timescales. }, journal = {Cell}, volume = {175}, year = {2018}, month = {2018 Nov 15}, pages = {1430-42}, abstract = {In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.}, doi = {10.1016/j.cell.2018.09.057}, author = {Guo, Yuting and Li, Di and Zhang, Siwei and Yang, Yanrui and Liu, Jia-Jia and Wang, Xinyu and Liu, Chong and Milkie, Daniel E and Moore, Regan P and Tulu, U Serdar and Kiehart, Daniel P and Hu, Junjie and Lippincott-Schwartz, Jennifer and Betzig, Eric and Li, Dong} } @article {49058, title = {VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.}, journal = {Scientific Reports}, volume = {8}, year = {2018}, month = {2018 Feb 20}, pages = {3353}, abstract = {

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4, which can{\textquoteright}t hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer{\textquoteright}s vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-21491-x}, author = {Ott, Carolyn and Nachmias, Dikla and Adar, Shai and Jarnik, Michal and Sherman, Shachar and Birnbaum, Ramon Y and Lippincott-Schwartz, Jennifer and Elia, Natalie} } @article {48415, title = {AMPK and vacuole-associated Atg14p orchestrate {\textmu}-lipophagy for energy production and long-term survival under glucose starvation.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Apr 10}, pages = {e21690}, abstract = {

Dietary restriction increases the longevity of many organisms but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy ({\textmu}-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger {\textmu}-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interacted with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating {\textmu}-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate {\textmu}-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.

}, issn = {2050-084X}, doi = {10.7554/eLife.21690}, author = {Seo, Arnold Young and Lau, Pick-Wei and Feliciano, Daniel and Sengupta, Prabuddha and Le Gros, Mark A and Cinquin, Bertrand and Larabell, Carolyn A and Lippincott-Schwartz, Jennifer} } @article {48534, title = {Applying systems-level spectral imaging and analysis to reveal the organelle interactome.}, journal = {Nature}, year = {2017}, month = {2017 May 24}, abstract = {

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

}, issn = {1476-4687}, doi = {10.1038/nature22369}, author = {Valm, Alex M and Cohen, Sarah and Legant, Wesley R and Melunis, Justin and Hershberg, Uri and Wait, Eric and Cohen, Andrew R and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {48858, title = {Cell volume change through water efflux impacts cell stiffness and stem cell fate.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Sep 25}, pages = {E8618-27}, abstract = {

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

}, issn = {1091-6490}, doi = {10.1073/pnas.1705179114}, author = {Guo, Ming and Pegoraro, Adrian F and Mao, Angelo and Zhou, Enhua H and Arany, Praveen R and Han, Yulong and Burnette, Dylan T and Jensen, Mikkel H and Kasza, Karen E and Moore, Jeffrey R and Mackintosh, Frederick C and Fredberg, Jeffrey J and Mooney, David J and Lippincott-Schwartz, Jennifer and Weitz, David A} } @article {48729, title = {Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Aug 08}, pages = {E6585-94}, abstract = {

CD8(+) cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.

}, issn = {1091-6490}, doi = {10.1073/pnas.1710751114}, author = {Ritter, Alex T and Kapnick, Senta M and Murugesan, Sricharan and Schwartzberg, Pamela L and Griffiths, Gillian M and Lippincott-Schwartz, Jennifer} } @article {48419, title = {Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.}, journal = {The Journal of Cell Biology}, volume = {216}, year = {2017}, month = {2017 Apr 07}, pages = {1337-55}, abstract = {

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

}, issn = {1540-8140}, doi = {10.1083/jcb.201609033}, author = {Allison, Rachel and Edgar, James R and Pearson, Guy and Rizo, Tania and Newton, Timothy and G{\"u}nther, Sven and Berner, Fiamma and Hague, Jennifer and Connell, James W and Winkler, J{\"u}rgen and Lippincott-Schwartz, Jennifer and Beetz, Christian and Winner, Beate and Reid, Evan} } @article {48939, title = {Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Nov 07}, pages = {E10056-65}, abstract = {

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

}, issn = {1091-6490}, doi = {10.1073/pnas.1706600114}, author = {Pak, Alexander J and Grime, John M A and Sengupta, Prabuddha and Chen, Antony K and Durumeric, Aleksander E P and Srivastava, Anand and Yeager, Mark and Briggs, John A G and Lippincott-Schwartz, Jennifer and Voth, Gregory A} } @article {48906, title = {Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.}, journal = {BMC Biology}, volume = {15}, year = {2017}, month = {2017 Oct 31}, pages = {102}, abstract = {

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth.

}, issn = {1741-7007}, doi = {10.1186/s12915-017-0432-0}, author = {Stefan, Christopher J and Trimble, William S and Grinstein, Sergio and Drin, Guillaume and Reinisch, Karin and De Camilli, Pietro and Cohen, Sarah and Valm, Alex M and Lippincott-Schwartz, Jennifer and Levine, Tim P and Iaea, David B and Maxfield, Frederick R and Futter, Clare E and Eden, Emily R and Judith, Delphine and van Vliet, Alexander R and Agostinis, Patrizia and Tooze, Sharon A and Sugiura, Ayumu and McBride, Heidi M} } @article {49352, title = {Myosin VI facilitates connexin 43 gap junction accretion.}, journal = {Journal of Cell Science}, volume = {130}, year = {2017}, month = {2017 03 01}, pages = {827-840}, abstract = {

In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI.

}, keywords = {Amino Acid Motifs, Animals, Cell Communication, Cell Membrane, Cells, Cultured, Connexin 43, Embryo, Mammalian, Fibroblasts, Gap Junctions, Golgi Apparatus, Hela Cells, Humans, Mice, Models, Biological, Myocardium, Myocytes, Cardiac, Myosin Heavy Chains, Protein Domains, Protein Transport, Rats}, issn = {1477-9137}, doi = {10.1242/jcs.199083}, author = {Waxse, Bennett J and Sengupta, Prabuddha and Hesketh, Geoffrey G and Lippincott-Schwartz, Jennifer and Buss, Folma} } @article {48650, title = {Rational engineering of photoconvertible fluorescent proteins for dual-color fluorescence nanoscopy enabled by a triplet-state mechanism of primed conversion.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {56}, year = {2017}, month = {2017 Jun 29}, pages = {11628-33}, abstract = {

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

}, issn = {1521-3773}, doi = {10.1002/anie.201706121}, author = {Mohr, Manuel Alexander and Kobitski, Andrei Yu and Sabater, Lluc Rullan and Nienhaus, Karin and Obara, Christopher John and Lippincott-Schwartz, Jennifer and Nienhaus, Gerd Ulrich and Pantazis, Periklis} } @article {47994, title = {AMPK activation prevents and reverses drug-induced mitochondrial and hepatocyte injury by promoting mitochondrial fusion and function.}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016 Oct 31}, pages = {e0165638}, abstract = {

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0165638}, author = {Kang, Sun Woo Sophie and Haydar, Ghada and Taniane, Caitlin and Farrell, Geoffrey and Arias, Irwin M and Lippincott-Schwartz, Jennifer and Fu, Dong} } @article {47729, title = {Bright photoactivatable fluorophores for single-molecule imaging.}, journal = {Nature Methods}, volume = {13}, year = {2016}, month = {2016 Oct 24}, pages = {985-8}, abstract = {

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

}, doi = {10.1038/nmeth.4034}, author = {Lavis, Luke D and Grimm, Jonathan B and English, Brian P and Choi, Heejin and Muthusamy, Anand K and Mehl, Brian P and Dong, Peng and Brown, Timothy A and Lippincott-Schwartz, Jennifer and Liu, Zhe and Lionnet, Timoth{\'e}e} } @article {47969, title = {Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.}, journal = {Science (New York, N.Y.)}, volume = {354}, year = {2016}, month = {2016 Oct 28}, pages = {433-46}, abstract = {

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER {\textquotedblleft}sheet{\textquotedblright} proliferation after overexpression of tubular junction{\textendash}forming proteins.

}, doi = {10.1126/science.aaf3928}, author = {Nixon-Abell, Jonathon and Obara, Christopher J and Weigel, Aubrey V and Li, Dong and Legant, Wesley R and Xu, CShan and Pasolli, H Amalia and Harvey, Kirsten and Hess, Harald F and Betzig, Eric and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {48135, title = {Live cell single molecule-guided Bayesian localization super resolution microscopy.}, journal = {Cell Research}, year = {2016}, month = {2016 Dec 30}, issn = {1748-7838}, doi = {10.1038/cr.2015.160}, author = {Xu, Fan and Zhang, Mingshu and He, Wenting and Han, Renmin and Xue, Fudong and Liu, Zhiyong and Zhang, Fa and Lippincott-Schwartz, Jennifer and Xu, Pingyong} } @article {48131, title = {The nanoscale spatial organization of B cell receptors on IgM- and IgG-expressing human B cells.}, journal = {Molecular Biology of the Cell}, volume = {28}, year = {2016}, month = {2016 Dec 14}, pages = {511-23}, abstract = {

B cell activation is initiated by the binding of antigen to the B cell receptor (BCR). Here we used dSTORM super resolution imaging to characterize the nanoscale spatial organization of IgM and IgG BCRs on the surfaces of resting and antigen-activated human peripheral blood B cells. We provide insights into both the fundamental process of antigen-driven BCR clustering as well as differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of na{\"\i}ve and memory B cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in both size and number of BCR single molecule localizations, both resting and activated B cells intrinsically maintain a high frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands following antigen activation. Small dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions.

}, issn = {1939-4586}, doi = {10.1091/mbc.E16-06-0452}, author = {Lee, Jinmin and Sengupta, Prabuddha and Brzostowski, Joseph and Lippincott-Schwartz, Jennifer and Pierce, Susan K} } @article {48116, title = {Sonic Hedgehog Pathway activation increases mitochondrial abundance and activity in hippocampal neurons.}, journal = {Molecular Biology of the Cell}, year = {2016}, month = {2016 Dec 08}, abstract = {

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. In this study we present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway impacts multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively, our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

}, issn = {1939-4586}, doi = {10.1091/mbc.E16-07-0553}, author = {Yao, Pamela J and Manor, Uri and Petralia, Ronald S and Brose, Rebecca D and Wu, Ryan T Y and Ott, Carolyn and Wang, Ya-Xian and Charnoff, Ari and Lippincott-Schwartz, Jennifer and Mattson, Mark P} } @article {46774, title = {Actin depletion initiates events leading to granule secretion at the immunological synapse.}, journal = {Immunity}, volume = {42}, year = {2015}, month = {2015 May 19}, pages = {864-76}, abstract = {

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1\ min, cortical actin reduced across the synapse, T\ cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5\ min and reached the synapse after 6\ min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

}, issn = {1097-4180}, doi = {10.1016/j.immuni.2015.04.013}, author = {Ritter, Alex T and Asano, Yukako and Stinchcombe, Jane C and Dieckmann, N M G and Chen, Bi-Chang and Gawden-Bone, C and van Engelenburg, Schuyler and Legant, Wesley and Gao, Liang and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer and Griffiths, Gillian M} } @article {47620, title = {Deacetylation of nuclear LC3 drives autophagy initiation under starvation.}, journal = {Molecular cell}, volume = {57}, year = {2015}, month = {2015 Feb 5}, pages = {456-66}, abstract = {

Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3{\textquoteright}s distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients.

}, keywords = {Acetylation, Autophagy, Cell Nucleus, Cytoplasm, HEK293 Cells, Hela Cells, Humans, Lysine, Microscopy, Confocal, Microtubule-Associated Proteins, Sirtuin 1, Ubiquitin-Activating Enzymes}, issn = {1097-4164}, doi = {10.1016/j.molcel.2014.12.013}, author = {Huang, Rui and Xu, Yinfeng and Wan, Wei and Shou, Xin and Qian, Jiali and You, Zhiyuan and Liu, Bo and Chang, Chunmei and Zhou, Tianhua and Lippincott-Schwartz, Jennifer and Liu, Wei} } @article {47618, title = {Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo.}, journal = {Biology open}, volume = {4}, year = {2015}, month = {2015}, pages = {301-11}, abstract = {

The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

}, issn = {2046-6390}, doi = {10.1242/bio.20149936}, author = {Rikhy, Richa and Mavrakis, Manos and Lippincott-Schwartz, Jennifer} } @article {47467, title = {Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.}, journal = {Nature Cell Biology}, volume = {17}, year = {2015}, month = {2015 Feb 16}, pages = {228-40}, abstract = {

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11{\textendash}​Rab8\ cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins ​EHD1\ and ​EHD3, in association with the Rab11{\textendash}​Rab8\ cascade, function in early ciliogenesis. ​EHD1\ and ​EHD3\ localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and ​IFT20. ​SNAP29, a SNARE membrane fusion regulator and ​EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is ​Rab8\ activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby ​EHD1\ and ​EHD3\ reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

}, issn = {1476-4679}, doi = {10.1038/ncb3109}, author = {Lu, Quanlong and Insinna, Christine and Ott, Carolyn and Stauffer, Jimmy and Pintado, Petra A and Rahajeng, Juliati and Baxa, Ulrich and Walia, Vijay and Cuenca, Adrian and Hwang, Yoo-Seok and Daar, Ira O and Lopes, Susana and Lippincott-Schwartz, Jennifer and Jackson, Peter K and Caplan, Steve and Westlake, Christopher J} } @article {47466, title = {Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.}, journal = {Developmental Cell}, volume = {32}, year = {2015}, month = {2015 Mar 23}, pages = {678-92}, abstract = {

Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells.

}, keywords = {Animals, Autophagy, Biological Transport, Cell Line, Fatty Acids, Fibroblasts, Fluorescent Dyes, Lipase, Lipid Droplets, Lipid Metabolism, Lipolysis, Mice, Mitochondria, Mitochondrial Dynamics, Oxidation-Reduction, Respiration, Starvation}, issn = {1878-1551}, doi = {10.1016/j.devcel.2015.01.029}, author = {Rambold, Angelika S and Cohen, Sarah and Lippincott-Schwartz, Jennifer} } @article {47619, title = {Intracellular and extracellular forces drive primary cilia movement.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Feb 3}, pages = {1410-5}, abstract = {

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

}, keywords = {Animals, Centrosome, Cilia, Dogs, Madin Darby Canine Kidney Cells, Microscopy, Electron, Movement}, issn = {1091-6490}, doi = {10.1073/pnas.1421845112}, author = {Battle, Christopher and Ott, Carolyn M and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Schmidt, Christoph F} } @article {47616, title = {Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Mar 3}, pages = {2630-2}, issn = {1091-6490}, doi = {10.1073/pnas.1500784112}, author = {Lippincott-Schwartz, Jennifer} } @article {47615, title = {Selective visualization of GLUT4 storage vesicles and associated Rab proteins using IRAP-pHluorin.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {1298}, year = {2015}, month = {2015}, pages = {173-9}, abstract = {

Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins.

}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Cattle, Cell Membrane, Cystinyl Aminopeptidase, Exocytosis, Glucose Transporter Type 4, Green Fluorescent Proteins, Image Processing, Computer-Assisted, Insulin, Mice, Microscopy, Fluorescence, rab GTP-Binding Proteins, Transfection}, issn = {1940-6029}, doi = {10.1007/978-1-4939-2569-8_14}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47621, title = {Cell Biology. Fixing problems with cell lines.}, journal = {Science (New York, N.Y.)}, volume = {346}, year = {2014}, month = {2014 Dec 19}, pages = {1452-3}, keywords = {Animals, Biological Specimen Banks, Biomedical Research, Cell Line, Drug Evaluation, Preclinical, Humans, National Institutes of Health (U.S.), Policy, Reproducibility of Results, United States}, issn = {1095-9203}, doi = {10.1126/science.1259110}, author = {Lorsch, Jon R and Collins, Francis S and Lippincott-Schwartz, Jennifer} } @article {47629, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {The Journal of cell biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {45153, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {Journal of Cell Biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {6710, title = {Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.}, journal = {Science}, volume = {343}, year = {2014}, month = {2014 Jan 16}, pages = {653-6}, abstract = {

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

}, issn = {1095-9203}, doi = {10.1126/science.1247786}, author = {Van Engelenburg, Schuyler B and Shtengel, Gleb and Sengupta, Prabuddha and Waki, Kayoko and Jarnik, Michal and Ablan, Sherimay D and Freed, Eric O and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {47631, title = {Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.}, journal = {Science (New York, N.Y.)}, volume = {343}, year = {2014}, month = {2014 Feb 7}, pages = {653-6}, abstract = {

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

}, keywords = {Endosomal Sorting Complexes Required for Transport, gag Gene Products, Human Immunodeficiency Virus, HIV Infections, HIV-1, Humans, Imaging, Three-Dimensional, Microscopy, Protein Subunits, Virion, Virus Assembly, Virus Release}, issn = {1095-9203}, doi = {10.1126/science.1247786}, author = {Van Engelenburg, Schuyler B and Shtengel, Gleb and Sengupta, Prabuddha and Waki, Kayoko and Jarnik, Michal and Ablan, Sherimay D and Freed, Eric O and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {47626, title = {ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory pathway.}, journal = {Cell}, volume = {158}, year = {2014}, month = {2014 Jul 31}, pages = {522-33}, abstract = {

Proteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. Here, we report that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.

}, keywords = {Animals, Cell Line, Endoplasmic Reticulum Stress, Humans, Lysosomes, Mice, Prions, Protein Folding, Rats, Secretory Pathway, Unfolded Protein Response}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.06.026}, author = {Satpute-Krishnan, Prasanna and Ajinkya, Monica and Bhat, Savithri and Itakura, Eisuke and Hegde, Ramanujan S and Lippincott-Schwartz, Jennifer} } @article {47624, title = {Flat clathrin lattices: stable features of the plasma membrane.}, journal = {Molecular biology of the cell}, volume = {25}, year = {2014}, month = {2014 Nov 5}, pages = {3581-94}, abstract = {

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

}, keywords = {Animals, Chemokine CCL5, CHO Cells, Clathrin, Clathrin-Coated Vesicles, Coated Pits, Cell-Membrane, Cricetulus, Dynamins, Endocytosis, Genes, Reporter, Green Fluorescent Proteins, HEK293 Cells, Hela Cells, Humans, Microscopy, Electron, Microscopy, Fluorescence, Molecular Imaging, Protein Transport, Receptors, CCR5}, issn = {1939-4586}, doi = {10.1091/mbc.E14-06-1154}, author = {Grove, Joe and Metcalf, Daniel J and Knight, Alex E and Wavre-Shapton, Sil{\`e}ne T and Sun, Tony and Protonotarios, Emmanouil D and Griffin, Lewis D and Lippincott-Schwartz, Jennifer and Marsh, Mark} } @article {45617, title = {Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {1257998}, abstract = {

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

}, issn = {1095-9203}, doi = {10.1126/science.1257998}, author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao, Lin and Milkie, Daniel E and Davidson, Michael W and Janetopoulos, Chris and Wu, Xufeng S and Hammer, John A and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz, Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and Mitchell, Diana M and Bembenek, Joshua N and Reymann, Anne-Cecile and B{\"o}hme, Ralph and Grill, Stephan W and Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and Kiehart, Daniel P and Betzig, Eric} } @article {47630, title = {LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.}, journal = {PloS one}, volume = {9}, year = {2014}, month = {2014}, pages = {e91921}, abstract = {

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

}, keywords = {AMP-Activated Protein Kinases, Animals, ATP-Binding Cassette Transporters, Bile Acids and Salts, Cell Membrane, Cell Polarity, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Endosomes, Gene Expression Regulation, Golgi Apparatus, Hepatocytes, Mice, Mice, Knockout, Myosin Type V, Primary Cell Culture, Protein Transport, Protein-Serine-Threonine Kinases, rab GTP-Binding Proteins, Signal Transduction, Taurocholic Acid}, issn = {1932-6203}, doi = {10.1371/journal.pone.0091921}, author = {Homolya, L{\'a}szl{\'o} and Fu, Dong and Sengupta, Prabuddha and Jarnik, Michal and Gillet, Jean-Pierre and Vitale-Cross, Lynn and Gutkind, J Silvio and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {47627, title = {MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, year = {2014}, month = {2014 Jul 1}, pages = {E2676-83}, abstract = {

MicroRNAs (miRNAs) are small, 18-22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA-protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA-Gag complexes interfere with viral-RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA{\textquoteright}s target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.

}, keywords = {Argonaute Proteins, Cluster Analysis, DNA Primers, HEK293 Cells, HIV-1, Humans, MicroRNAs, Microscopy, Fluorescence, Multiprotein Complexes, Protein Precursors, RNA, Small Interfering, Virus Assembly, Virus Release}, issn = {1091-6490}, doi = {10.1073/pnas.1408037111}, author = {Chen, Antony K and Sengupta, Prabuddha and Waki, Kayoko and Van Engelenburg, Schuyler B and Ochiya, Takahiro and Ablan, Sherimay D and Freed, Eric O and Lippincott-Schwartz, Jennifer} } @article {47628, title = {Photocontrollable fluorescent proteins for superresolution imaging.}, journal = {Annual review of biophysics}, volume = {43}, year = {2014}, month = {2014}, pages = {303-29}, abstract = {

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.

}, keywords = {Animals, Cells, Luminescent Proteins, Mammals, Microscopy, Fluorescence}, issn = {1936-1238}, doi = {10.1146/annurev-biophys-051013-022836}, author = {Shcherbakova, Daria M and Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer and Verkhusha, Vladislav V} } @article {47625, title = {Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy.}, journal = {Cell}, volume = {158}, year = {2014}, month = {2014 Aug 14}, pages = {822-32}, abstract = {

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force\ fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

}, keywords = {Animals, Biomechanical Phenomena, Cytoplasm, Embryo, Mammalian, Fibroblasts, Mice, Microscopy, Atomic Force, Proteins, Vimentin}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.06.051}, author = {Guo, Ming and Ehrlicher, Allen J and Jensen, Mikkel H and Renz, Malte and Moore, Jeffrey R and Goldman, Robert D and Lippincott-Schwartz, Jennifer and Mackintosh, Frederick C and Weitz, David A} } @article {47622, title = {Quantitative cell biology: transforming the conceptual, theoretical, instrumental, and methodological approaches to cell biology.}, journal = {Molecular biology of the cell}, volume = {25}, year = {2014}, month = {2014 Nov 5}, pages = {3437}, keywords = {Animals, Cell Biology, Computer Simulation, Humans, Models, Biological, Molecular Imaging}, issn = {1939-4586}, doi = {10.1091/mbc.E14-08-1297}, author = {Lippincott-Schwartz, Jennifer} } @article {47632, title = {Superresolution imaging of biological systems using photoactivated localization microscopy.}, journal = {Chemical reviews}, volume = {114}, year = {2014}, month = {2014 Mar 26}, pages = {3189-202}, keywords = {Image Processing, Computer-Assisted, Luminescent Proteins, Microscopy, Mitochondria, Nanotechnology, Time-Lapse Imaging}, issn = {1520-6890}, doi = {10.1021/cr400614m}, author = {Sengupta, Prabuddha and Van Engelenburg, Schuyler B and Lippincott-Schwartz, Jennifer} } @article {47623, title = {Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.}, journal = {Molecular biology of the cell}, volume = {25}, year = {2014}, month = {2014 Dec 15}, pages = {4166-73}, abstract = {

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure-function relationship of EB1 by generating EB1-PACF dimers (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.

}, keywords = {Amino Acid Sequence, Cell Movement, Hela Cells, Humans, MCF-7 Cells, Microscopy, Fluorescence, Microtubule-Associated Proteins, Microtubules, Molecular Sequence Data, Protein Transport}, issn = {1939-4586}, doi = {10.1091/mbc.E14-06-1133}, author = {Xia, Peng and Liu, Xing and Wu, Bing and Zhang, Shuyuan and Song, Xiaoyu and Yao, Phil Y and Lippincott-Schwartz, Jennifer and Yao, Xuebiao} } @article {47642, title = {Accelerating 3B single-molecule super-resolution microscopy with cloud computing.}, journal = {Nature methods}, volume = {10}, year = {2013}, month = {2013 Feb}, pages = {96-7}, keywords = {Computational Biology, Computers, Fluorescent Dyes, Microscopy, Fluorescence, Nanotechnology}, issn = {1548-7105}, doi = {10.1038/nmeth.2335}, author = {Hu, Ying S and Nan, Xiaolin and Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer and Cang, Hu} } @article {47640, title = {Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Apr 30}, pages = {7288-93}, abstract = {

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

}, keywords = {Adenosine Triphosphate, Adenylate Kinase, Animals, Autophagy, Cell Polarity, Energy Metabolism, Fatty Acids, Glycolysis, Hepatocytes, Lipids, Membrane Potential, Mitochondrial, Mitochondria, Mitochondrial Dynamics, Mitochondrial Proteins, Oxidation-Reduction, Oxidative Phosphorylation, Rats, Up-Regulation}, issn = {1091-6490}, doi = {10.1073/pnas.1304285110}, author = {Fu, Dong and Mitra, Kasturi and Sengupta, Prabuddha and Jarnik, Michal and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {6669, title = {Fast structural responses of gap junction membrane domains to AB5 toxins.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Oct 29}, pages = {E4125-33}, abstract = {

Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast (\ 500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

}, issn = {1091-6490}, doi = {10.1073/pnas.1315850110}, author = {Majoul, Irina V and Gao, Liang and Betzig, Eric and Onichtchouk, Daria and Butkevich, Eugenia and Kozlov, Yuri and Bukauskas, Feliksas and Bennett, Michael V L and Lippincott-Schwartz, Jennifer and Duden, Rainer} } @article {47637, title = {Fast structural responses of gap junction membrane domains to AB5 toxins.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Oct 29}, pages = {E4125-33}, abstract = {

Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast (~500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

}, keywords = {Analysis of Variance, Animals, Bacterial Toxins, Bicyclo Compounds, Heterocyclic, Cercopithecus aethiops, Connexins, Cyclic AMP, DNA Primers, Filipin, Fluorescence, Gap Junctions, Image Processing, Computer-Assisted, Membrane Lipids, Microscopy, Confocal, Patch-Clamp Techniques, Potassium, Thiazolidines, Vero Cells}, issn = {1091-6490}, doi = {10.1073/pnas.1315850110}, author = {Majoul, Irina V and Gao, Liang and Betzig, Eric and Onichtchouk, Daria and Butkevich, Eugenia and Kozlov, Yuri and Bukauskas, Feliksas and Bennett, Michael V L and Lippincott-Schwartz, Jennifer and Duden, Rainer} } @article {47634, title = {Incisive imaging and computation for cellular mysteries: lessons from abscission.}, journal = {Cell}, volume = {155}, year = {2013}, month = {2013 Dec 5}, pages = {1220-31}, abstract = {

The final cleavage event that terminates cell division, abscission of the small, dense intercellular bridge, has been particularly challenging to resolve. Here, we describe imaging innovations that helped answer long-standing questions about the mechanism of abscission. We further explain how computational modeling of high-resolution data was employed to test hypotheses and generate additional insights. We present the model that emerges from application of these complimentary approaches. Similar experimental strategies will undoubtedly reveal exciting details about other underresolved cellular structures.

}, keywords = {Animals, Cell Physiological Processes, Cytokinesis, Humans, Models, Biological, Plant Cells}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.11.011}, author = {Elia, Natalie and Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47636, title = {Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures.}, journal = {Autophagy}, volume = {9}, year = {2013}, month = {2013 Dec}, pages = {2154-5}, abstract = {

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for ?-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.

}, keywords = {Animals, Autophagy, Cell Culture Techniques, Cell Polarity, Cell Separation, Cells, Cultured, Collagen, Hepatocytes, Mitochondrial Dynamics, Primary Cell Culture, Rats, Tissue Scaffolds}, issn = {1554-8635}, doi = {10.4161/auto.26167}, author = {Fu, Dong and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {47638, title = {Insulin triggers surface-directed trafficking of sequestered GLUT4 storage vesicles marked by Rab10.}, journal = {Small GTPases}, volume = {4}, year = {2013}, month = {2013 Jul-Sep}, pages = {193-7}, abstract = {

Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don{\textquoteright}t interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

}, keywords = {Adipocytes, Animals, Cell Line, Cell Membrane, Endosomes, Glucose Transporter Type 4, Insulin, Mice, rab GTP-Binding Proteins, RNA Interference, RNA, Small Interfering}, issn = {2154-1256}, doi = {10.4161/sgtp.26471}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {6130, title = {Nanotools for neuroscience and brain activity mapping.}, journal = {ACS Nano}, volume = {7}, year = {2013}, month = {2013 Mar 26}, pages = {1850-66}, abstract = {

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

}, issn = {1936-086X}, doi = {10.1021/nn4012847}, author = {Alivisatos, A Paul and Andrews, Anne M and Boyden, Edward S and Chun, Miyoung and Church, George M and Deisseroth, Karl and Donoghue, John P and Fraser, Scott E and Lippincott-Schwartz, Jennifer and Looger, Loren L and Masmanidis, Sotiris and McEuen, Paul L and Nurmikko, Arto V and Park, Hongkun and Peterka, Darcy S and Reid, Clay and Roukes, Michael L and Scherer, Axel and Schnitzer, Mark and Sejnowski, Terrence J and Shepard, Kenneth L and Tsao, Doris and Turrigiano, Gina and Weiss, Paul S and Xu, Chris and Yuste, Rafael and Zhuang, Xiaowei} } @article {47641, title = {Nanotools for neuroscience and brain activity mapping.}, journal = {ACS nano}, volume = {7}, year = {2013}, month = {2013 Mar 26}, pages = {1850-66}, abstract = {

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

}, keywords = {Animals, Brain Mapping, Humans, Models, Neurological, Nanomedicine, Nanoparticles, Nanotechnology, Nervous System Physiological Phenomena}, issn = {1936-086X}, doi = {10.1021/nn4012847}, author = {Alivisatos, A Paul and Andrews, Anne M and Boyden, Edward S and Chun, Miyoung and Church, George M and Deisseroth, Karl and Donoghue, John P and Fraser, Scott E and Lippincott-Schwartz, Jennifer and Looger, Loren L and Masmanidis, Sotiris and McEuen, Paul L and Nurmikko, Arto V and Park, Hongkun and Peterka, Darcy S and Reid, Clay and Roukes, Michael L and Scherer, Axel and Schnitzer, Mark and Sejnowski, Terrence J and Shepard, Kenneth L and Tsao, Doris and Turrigiano, Gina and Weiss, Paul S and Xu, Chris and Yuste, Rafael and Zhuang, Xiaowei} } @article {47644, title = {NBR1 acts as an autophagy receptor for peroxisomes.}, journal = {Journal of cell science}, volume = {126}, year = {2013}, month = {2013 Feb 15}, pages = {939-52}, abstract = {

Selective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62, NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates to nascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes (pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that the amphipathic α-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain are necessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J and UBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.

}, keywords = {Autophagy, Blotting, Western, Cell Line, Hela Cells, Humans, Microscopy, Confocal, Microscopy, Electron, Peroxisomes, Proteins}, issn = {1477-9137}, doi = {10.1242/jcs.114819}, author = {Deosaran, Elizabeth and Larsen, Kenneth B and Hua, Rong and Sargent, Graeme and Wang, Yuqing and Kim, Sarah and Lamark, Trond and Jauregui, Miluska and Law, Kelsey and Lippincott-Schwartz, Jennifer and Brech, Andreas and Johansen, Terje and Kim, Peter K} } @article {47635, title = {Photohighlighting approaches to access membrane dynamics of the Golgi apparatus.}, journal = {Methods in cell biology}, volume = {118}, year = {2013}, month = {2013}, pages = {217-34}, abstract = {

By providing quantitative, visual data of live cells, fluorescent protein-based microscopy techniques are furnishing novel insights into the complexities of membrane trafficking pathways and organelle dynamics. In this chapter, we describe experimental protocols employing fluorescent protein-based photohighlighting techniques to quantify protein movement into and out of the Golgi apparatus, an organelle that serves as the central sorting and processing station of the secretory pathway. The methods allow kinetic characteristics of Golgi-associated protein trafficking to be deciphered, which can help clarify how the Golgi maintains itself as a steady-state structure despite a continuous flux of secretory cargo passing into and out of this organelle. The guidelines presented in this chapter can also be applied to examine the dynamics of other intracellular organelle systems, elucidating mechanisms for how proteins are maintained in specific organelles and/or circulated to other destinations within the cell.

}, keywords = {Animals, Cercopithecus aethiops, CHO Cells, COS Cells, Cricetinae, Cricetulus, Fluorescence Recovery After Photobleaching, Golgi Apparatus, Green Fluorescent Proteins, Hela Cells, Humans, Membrane Glycoproteins, Protein Transport, Recombinant Fusion Proteins, Secretory Pathway, Viral Envelope Proteins}, issn = {0091-679X}, doi = {10.1016/B978-0-12-417164-0.00013-6}, author = {Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer} } @article {47643, title = {Quantifying spatial organization in point-localization superresolution images using pair correlation analysis.}, journal = {Nature protocols}, volume = {8}, year = {2013}, month = {2013 Feb}, pages = {345-54}, abstract = {

The distinctive distributions of proteins within subcellular compartments both at steady state and during signaling events have essential roles in cell function. Here we describe a method for delineating the complex arrangement of proteins within subcellular structures visualized using point-localization superresolution (PL-SR) imaging. The approach, called pair correlation photoactivated localization microscopy (PC-PALM), uses a pair-correlation algorithm to precisely identify single molecules in PL-SR imaging data sets, and it is used to decipher quantitative features of protein organization within subcellular compartments, including the existence of protein clusters and the size, density and number of proteins in these clusters. We provide a step-by-step protocol for PC-PALM, illustrating its analysis capability for four plasma membrane proteins tagged with photoactivatable GFP (PAGFP). The experimental steps for PC-PALM can be carried out in 3 d and the analysis can be done in \~{}6-8 h. Researchers need to have substantial experience in single-molecule imaging and statistical analysis to conduct the experiments and carry out this analysis.

}, keywords = {Algorithms, Green Fluorescent Proteins, Image Processing, Computer-Assisted, Intracellular Space, Membrane Proteins, Microscopy}, issn = {1750-2799}, doi = {10.1038/nprot.2013.005}, author = {Sengupta, Prabuddha and Jovanovic-Talisman, Tijana and Lippincott-Schwartz, Jennifer} } @article {47639, title = {Rab10 delivers GLUT4 storage vesicles to the plasma membrane.}, journal = {Communicative \& integrative biology}, volume = {6}, year = {2013}, month = {2013 May 1}, pages = {e23779}, abstract = {

The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.

}, issn = {1942-0889}, doi = {10.4161/cib.23779}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47633, title = {Superresolution imaging with standard fluorescent probes.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {60}, year = {2013}, month = {2013}, pages = {Unit 21.8.}, abstract = {

For more than 100 years, the ultimate resolution of a light microscope (\~{} 200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today{\textquoteright}s light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access information at an order of magnitude greater resolution than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but, as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking-assisted localization microscopy (BaLM) makes use of the inherent blinking and bleaching properties of almost all fluorophores as a means to generate superresolution images.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Microscopy, Fluorescence, Signal-To-Noise Ratio}, issn = {1934-2616}, doi = {10.1002/0471143030.cb2108s60}, author = {Millis, Bryan A and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Kachar, Bechara} } @article {47661, title = {Bayesian localization microscopy reveals nanoscale podosome dynamics.}, journal = {Nature methods}, volume = {9}, year = {2012}, month = {2012 Feb}, pages = {195-200}, abstract = {

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

}, keywords = {Bayes Theorem, Cell Line, Tumor, Humans, Nanotechnology}, issn = {1548-7105}, doi = {10.1038/nmeth.1812}, author = {Cox, Susan and Rosten, Edward and Monypenny, James and Jovanovic-Talisman, Tijana and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Jones, Gareth E and Heintzmann, Rainer} } @article {47652, title = {Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.}, journal = {Blood}, volume = {119}, year = {2012}, month = {2012 Feb 23}, pages = {1848-55}, abstract = {

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

}, keywords = {Animals, Antigens, CD34, Antigens, CD82, Bone Marrow, Cell Cycle, Cell Line, Tumor, Cells, Cultured, Cytokines, Flow Cytometry, Fluorescent Antibody Technique, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Humans, Interleukin Receptor Common gamma Subunit, Leukemia, Membrane Microdomains, Mice, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Microscopy, Confocal, Receptors, CXCR4, Tetraspanins, Transplantation, Heterologous}, issn = {1528-0020}, doi = {10.1182/blood-2011-08-371583}, author = {Larochelle, Andre and Gillette, Jennifer M and Desmond, Ronan and Ichwan, Brian and Cantilena, Amy and Cerf, Alexandra and Barrett, A John and Wayne, Alan S and Lippincott-Schwartz, Jennifer and Dunbar, Cynthia E} } @article {47659, title = {Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.}, journal = {Blood}, volume = {119}, year = {2012}, month = {2012 Feb 23}, pages = {1848-55}, abstract = {

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

}, keywords = {Animals, Antigens, CD34, Antigens, CD82, Bone Marrow, Cell Cycle, Cell Line, Tumor, Cells, Cultured, Cytokines, Flow Cytometry, Fluorescent Antibody Technique, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Humans, Interleukin Receptor Common gamma Subunit, Leukemia, Membrane Microdomains, Mice, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Microscopy, Confocal, Receptors, CXCR4, Tetraspanins, Transplantation, Heterologous}, issn = {1528-0020}, doi = {10.1182/blood-2011-08-371583}, author = {Larochelle, Andre and Gillette, Jennifer M and Desmond, Ronan and Ichwan, Brian and Cantilena, Amy and Cerf, Alexandra and Barrett, A John and Wayne, Alan S and Lippincott-Schwartz, Jennifer and Dunbar, Cynthia E} } @article {47649, title = {Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling.}, journal = {Biophysical journal}, volume = {102}, year = {2012}, month = {2012 May 16}, pages = {2309-20}, abstract = {

The endosomal sorting complex required for transport (ESCRT)-III complex, capable of polymerization and remodeling, participates in abscission of the intercellular membrane bridge connecting two daughter cells at the end of cytokinesis. Here, we integrate quantitative imaging of ESCRT-III during cytokinetic abscission with biophysical properties of ESCRT-III complexes to formulate and test a computational model for ESCRT-mediated cytokinetic abscission. We propose that cytokinetic abscission is driven by an ESCRT-III fission complex, which arises from ESCRT-III polymerization at the edge of the cytokinetic midbody structure, located at the center of the intercellular bridge. Formation of the fission complex is completed by remodeling and breakage of the ESCRT-III polymer assisted by VPS4. Subsequent spontaneous constriction of the fission complex generates bending deformation of the intercellular bridge membrane. The related membrane elastic force propels\ the fission complex along the intercellular bridge away from the midbody until it reaches an equilibrium position, determining the scission site. Membrane attachment to the dome-like end-cap of the fission complex drives membrane fission, completing the abscission process. We substantiate the model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions.

}, keywords = {Adenosine Triphosphate, Animals, Cell Line, Cell Membrane, Computer Simulation, Cytokinesis, Dogs, Endosomal Sorting Complexes Required for Transport, Extracellular Space, Humans, Models, Biological, Polymerization, Reproducibility of Results, Vacuolar Proton-Translocating ATPases}, issn = {1542-0086}, doi = {10.1016/j.bpj.2012.04.007}, author = {Elia, Natalie and Fabrikant, Gur and Kozlov, Michael M and Lippincott-Schwartz, Jennifer} } @article {47650, title = {DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis.}, journal = {The Journal of cell biology}, volume = {197}, year = {2012}, month = {2012 May 14}, pages = {487-97}, abstract = {

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. In this paper, we demonstrate a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1-depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis.

}, keywords = {Animals, Cell Cycle Proteins, Cell Differentiation, Cytoskeletal Proteins, Drosophila melanogaster, Drosophila Proteins, GTP-Binding Proteins, Mitochondria, Mitosis, Oogenesis, Receptors, Notch}, issn = {1540-8140}, doi = {10.1083/jcb.201110058}, author = {Mitra, Kasturi and Rikhy, Richa and Lilly, Mary and Lippincott-Schwartz, Jennifer} } @article {47653, title = {Intracellular spatial localization regulated by the microtubule network.}, journal = {PloS one}, volume = {7}, year = {2012}, month = {2012}, pages = {e34919}, abstract = {

The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a "structured cytoplasm", in contrast to a free and fluid environment.

}, keywords = {Animals, Cell Cycle, Centrosome, Cytoplasm, Drosophila, Microtubules, Models, Biological, Spindle Apparatus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0034919}, author = {Chen, Jing and Lippincott-Schwartz, Jennifer and Liu, Jian} } @article {47648, title = {Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 May 29}, pages = {8588-93}, abstract = {

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

}, keywords = {Algorithms, Animals, Animals, Genetically Modified, Blastoderm, Cell Compartmentation, Cell Membrane, Computer Simulation, Cytoplasm, Diffusion, Drosophila melanogaster, Embryo, Nonmammalian, Fluorescence Recovery After Photobleaching, Giant Cells, Green Fluorescent Proteins, Kinetics, Microscopy, Fluorescence, Mitosis, Models, Biological}, issn = {1091-6490}, doi = {10.1073/pnas.1204270109}, author = {Daniels, Brian R and Rikhy, Richa and Renz, Malte and Dobrowsky, Terrence M and Lippincott-Schwartz, Jennifer} } @article {47646, title = {Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Oct 30}, pages = {E2989-97}, abstract = {

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

}, keywords = {Asialoglycoprotein Receptor, Fluorescence Resonance Energy Transfer, Ligands, Microscopy, Confocal}, issn = {1091-6490}, doi = {10.1073/pnas.1211753109}, author = {Renz, Malte and Daniels, Brian R and V{\'a}mosi, Gy{\"o}rgy and Arias, Irwin M and Lippincott-Schwartz, Jennifer} } @article {47656, title = {Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Oct 30}, pages = {E2989-97}, abstract = {

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

}, keywords = {Asialoglycoprotein Receptor, Fluorescence Resonance Energy Transfer, Ligands, Microscopy, Confocal}, issn = {1091-6490}, doi = {10.1073/pnas.1211753109}, author = {Renz, Malte and Daniels, Brian R and V{\'a}mosi, Gy{\"o}rgy and Arias, Irwin M and Lippincott-Schwartz, Jennifer} } @article {47651, title = {Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair-correlation analysis.}, journal = {BioEssays : news and reviews in molecular, cellular and developmental biology}, volume = {34}, year = {2012}, month = {2012 May}, pages = {396-405}, abstract = {

Pointillistic based super-resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super-resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair-correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described.

}, keywords = {Algorithms, Animals, Cell Membrane, Cercopithecus aethiops, COS Cells, Green Fluorescent Proteins, Humans, Image Processing, Computer-Assisted, Microscopy, Fluorescence, Photons, Proteins}, issn = {1521-1878}, doi = {10.1002/bies.201200022}, author = {Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer} } @article {47647, title = {Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes.}, journal = {The Journal of cell biology}, volume = {198}, year = {2012}, month = {2012 Aug 20}, pages = {545-60}, abstract = {

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Cell Membrane, Cytoplasmic Vesicles, Glucose Transporter Type 4, Insulin, Mice, Myosin Heavy Chains, Myosin Type V, Protein Transport, rab GTP-Binding Proteins}, issn = {1540-8140}, doi = {10.1083/jcb.201111091}, author = {Chen, Yu and Wang, Yan and Zhang, Jinzhong and Deng, Yongqiang and Jiang, Li and Song, Eli and Wu, Xufeng S and Hammer, John A and Xu, Tao and Lippincott-Schwartz, Jennifer} } @article {47645, title = {Visualization of live primary cilia dynamics using fluorescence microscopy.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 4}, year = {2012}, month = {2012 Dec}, pages = {Unit 4.26}, abstract = {

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

}, keywords = {Animals, Bodily Secretions, Cilia, Dyneins, Epithelial Cells, Flagella, Humans, Kinesin, Kymography, Microscopy, Fluorescence, Microtubules, Photobleaching, Primary Cell Culture, Signal Transduction}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0426s57}, author = {Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47654, title = {Visualization of live primary cilia dynamics using fluorescence microscopy.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 4}, year = {2012}, month = {2012 Dec}, pages = {Unit 4.26}, abstract = {

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

}, keywords = {Animals, Bodily Secretions, Cilia, Dyneins, Epithelial Cells, Flagella, Humans, Kinesin, Kymography, Microscopy, Fluorescence, Microtubules, Photobleaching, Primary Cell Culture, Signal Transduction}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0426s57}, author = {Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47655, title = {Visualizing cell structure and function with point-localization superresolution imaging.}, journal = {Developmental cell}, volume = {23}, year = {2012}, month = {2012 Dec 11}, pages = {1092-102}, abstract = {

Fundamental to the success of cell and developmental biology is the ability to tease apart molecular organization in cells and tissues by localizing specific proteins with respect to one another in a native cellular context. However, many key cellular structures (from mitochondrial cristae to nuclear pores) lie below the diffraction limit of visible light, precluding analysis of their organization by conventional approaches. Point-localization superresolution microscopy techniques, such as PALM and STORM, are poised to resolve, with unprecedented clarity, the organizational principles of macromolecular complexes within cells, thus leading to deeper insights into cellular function in both health and disease.

}, keywords = {Cell Physiological Phenomena, Cells, Cellular Structures, Fluorescent Dyes, Imaging, Three-Dimensional, Microscopy, Microscopy, Fluorescence, Mitochondrial Membranes}, issn = {1878-1551}, doi = {10.1016/j.devcel.2012.09.022}, author = {Sengupta, Prabuddha and van Engelenburg, Schuyler and Lippincott-Schwartz, Jennifer} } @article {47674, title = {Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Jan 25}, pages = {1403-8}, abstract = {

This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.

}, keywords = {Adenylate Kinase, Animals, Bile Acids and Salts, Blotting, Western, Cell Polarity, Cells, Cultured, Chenodeoxycholic Acid, Cholagogues and Choleretics, Cyclic AMP, Dose-Response Relationship, Drug, Green Fluorescent Proteins, Guanine Nucleotide Exchange Factors, Hepatocytes, Imines, Microscopy, Confocal, Mitogen-Activated Protein Kinase Kinases, Models, Biological, Protein-Serine-Threonine Kinases, rap1 GTP-Binding Proteins, Rats, Rats, Sprague-Dawley, Signal Transduction, Taurocholic Acid, Ursodeoxycholic Acid}, issn = {1091-6490}, doi = {10.1073/pnas.1018376108}, author = {Fu, Dong and Wakabayashi, Yoshiyuki and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {47660, title = {Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Dec 27}, pages = {21081-6}, abstract = {

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Fluorescent Dyes, Green Fluorescent Proteins, Image Interpretation, Computer-Assisted, Microscopy, Fluorescence, Photobleaching, Software}, issn = {1091-6490}, doi = {10.1073/pnas.1117430109}, author = {Burnette, Dylan T and Sengupta, Prabuddha and Dai, Yuhai and Lippincott-Schwartz, Jennifer and Kachar, Bechara} } @article {47668, title = {Bridging structure and process in developmental biology through new imaging technologies.}, journal = {Developmental cell}, volume = {21}, year = {2011}, month = {2011 Jul 19}, pages = {5-10}, abstract = {

Many unexpected discoveries in developmental biology have depended on advancement of imaging technologies to visualize developmental processes as they unfold across multiple spatial and temporal scales. This essay surveys the recent advances in imaging, highlighting emerging capabilities with an eye toward those poised to have the greatest impact on developmental biology.

}, keywords = {Animals, Cell Biology, Cells, Developmental Biology, Green Fluorescent Proteins, Microscopy}, issn = {1878-1551}, doi = {10.1016/j.devcel.2011.06.030}, author = {Lippincott-Schwartz, Jennifer} } @article {47666, title = {Cell biology. SevERing mitochondria.}, journal = {Science (New York, N.Y.)}, volume = {334}, year = {2011}, month = {2011 Oct 14}, pages = {186-7}, keywords = {Cytosol, Dynamins, Endoplasmic Reticulum, Membrane Proteins, Mitochondria, Mitochondrial Proteins, Models, Biological, Yeasts}, issn = {1095-9203}, doi = {10.1126/science.1214059}, author = {Rambold, Angelika S and Lippincott-Schwartz, Jennifer} } @article {47657, title = {Cellular mechanism of bile acid-accelerated hepatocyte polarity.}, journal = {Small GTPases}, volume = {2}, year = {2011}, month = {2011 Nov 1}, pages = {314-317}, abstract = {

We recently discovered that the major mammalian bile acid, taurocholate, accelerated polarity in primary rat hepatocytes. Taurocholate increased cellular cAMP and signals through an Epac-Rap1-MEK-LKB1-AMPK pathway for its polarity effect. This review discusses possible mechanisms for how taurocholate affects different cell polarity factors, particularly AMPK, and thereby regulates events that generate polarity. These include tight junction formation, apical trafficking, recycling endosome dynamics, and cytoskeleton rearrangement. We also discuss whether the effects of taurocholate are mediated by other LKB1 downstream kinases, such as Par1 and NUAK1.

}, issn = {2154-1256}, doi = {10.4161/sgtp.18087}, author = {Fu, Dong and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {47672, title = {Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Mar 22}, pages = {4846-51}, abstract = {

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.

}, keywords = {Animals, Cell Line, Cytokinesis, DNA-Binding Proteins, Dogs, Endosomal Sorting Complexes Required for Transport, Endosomes, Humans, Transcription Factors}, issn = {1091-6490}, doi = {10.1073/pnas.1102714108}, author = {Elia, Natalie and Sougrat, Rachid and Spurlin, Tighe A and Hurley, James H and Lippincott-Schwartz, Jennifer} } @article {47670, title = {Emerging in vivo analyses of cell function using fluorescence imaging (*).}, journal = {Annual review of biochemistry}, volume = {80}, year = {2011}, month = {2011}, pages = {327-32}, abstract = {

Understanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.

}, keywords = {Cell Physiological Phenomena, Fluorescence Resonance Energy Transfer, Luminescent Proteins, Microscopy, Fluorescence, Nitric Oxide, Signal Transduction, Zinc}, issn = {1545-4509}, doi = {10.1146/annurev-biochem-121010-125553}, author = {Lippincott-Schwartz, Jennifer} } @article {47664, title = {An evolving paradigm for the secretory pathway?}, journal = {Molecular biology of the cell}, volume = {22}, year = {2011}, month = {2011 Nov}, pages = {3929-32}, abstract = {

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes.

}, keywords = {Animals, Awards and Prizes, Cells, Cultured, Fluorescence Recovery After Photobleaching, Green Fluorescent Proteins, Humans, Microscopy, Electron, Microscopy, Video, Models, Biological, Protein Transport, Proteins, Secretory Vesicles}, issn = {1939-4586}, doi = {10.1091/mbc.E11-05-0452}, author = {Lippincott-Schwartz, Jennifer} } @article {47663, title = {Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor.}, journal = {Immunity}, volume = {35}, year = {2011}, month = {2011 Nov 23}, pages = {705-20}, abstract = {

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

}, keywords = {Actins, Adaptor Proteins, Signal Transducing, Cell Line, Tumor, Cells, Cultured, Humans, Jurkat Cells, Lymphocyte Activation, Membrane Proteins, Phospholipase C gamma, Phosphoproteins, Phosphorylation, Protein Binding, Protein Multimerization, Protein Transport, Receptors, Antigen, T-Cell, Receptors, Antigen, T-Cell, gamma-delta, Signal Transduction, T-Lymphocytes, ZAP-70 Protein-Tyrosine Kinase}, issn = {1097-4180}, doi = {10.1016/j.immuni.2011.10.004}, author = {Sherman, Eilon and Barr, Valarie and Manley, Suliana and Patterson, George and Balagopalan, Lakshmi and Akpan, Itoro and Regan, Carole K and Merrill, Robert K and Sommers, Connie L and Lippincott-Schwartz, Jennifer and Samelson, Lawrence E} } @article {47658, title = {Fuse or die: Shaping mitochondrial fate during starvation.}, journal = {Communicative \& integrative biology}, volume = {4}, year = {2011}, month = {2011 Nov 1}, pages = {752-4}, abstract = {

Mitochondria continuously change their shape and thereby influence different cellular processes like cell death or development. Recently, we showed that during starvation mitochondria fuse into a highly connected network. The change in mitochondrial shape was dependent on inactivation of the fission protein Drp1, through targeting of two different phosphorylation sites. This rapid inhibition of mitochondrial fission led to unopposed fusion, protecting mitochondria from starvation-induced degradation and enabling the cell to survive nutrient scarce conditions.

}, issn = {1942-0889}, author = {Rambold, Angelika S and Kostelecky, Brenda and Lippincott-Schwartz, Jennifer} } @article {47662, title = {Mechanisms of mitochondria and autophagy crosstalk.}, journal = {Cell cycle (Georgetown, Tex.)}, volume = {10}, year = {2011}, month = {2011 Dec 1}, pages = {4032-8}, abstract = {

Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuronal loss in Parkinson{\textquoteright}s disease. In addition to autophagy{\textquoteright}s significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria{\textquoteright}s ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.

}, keywords = {Animals, Apoptosis, Autophagy, DNA Replication, DNA, Mitochondrial, Humans, Mammals, Metabolic Diseases, Microtubule-Associated Proteins, Mitochondria, Mitochondrial Membranes, Mitochondrial Proteins, Stress, Physiological}, issn = {1551-4005}, doi = {10.4161/cc.10.23.18384}, author = {Rambold, Angelika S and Lippincott-Schwartz, Jennifer} } @article {47673, title = {Principles and current strategies for targeting autophagy for cancer treatment.}, journal = {Clinical cancer research : an official journal of the American Association for Cancer Research}, volume = {17}, year = {2011}, month = {2011 Feb 15}, pages = {654-66}, abstract = {

Autophagy is an evolutionarily conserved, intracellular self-defense mechanism in which organelles and proteins are sequestered into autophagic vesicles that are subsequently degraded through fusion with lysosomes. Cells, thereby, prevent the toxic accumulation of damaged or unnecessary components, but also recycle these components to sustain metabolic homoeostasis. Heightened autophagy is a mechanism of resistance for cancer cells faced with metabolic and therapeutic stress, revealing opportunities for exploitation as a therapeutic target in cancer. We summarize recent developments in the field of autophagy and cancer and build upon the results presented at the Cancer Therapy Evaluation Program (CTEP) Early Drug Development meeting in March 2010. Herein, we describe our current understanding of the core components of the autophagy machinery and the functional relevance of autophagy within the tumor microenvironment, and we outline how this knowledge has informed preclinical investigations combining the autophagy inhibitor hydroxychloroquine (HCQ) with chemotherapy, targeted therapy, and immunotherapy. Finally, we describe ongoing clinical trials involving HCQ as a first generation autophagy inhibitor, as well as strategies for the development of novel, more potent, and specific inhibitors of autophagy.

}, keywords = {Animals, Antineoplastic Agents, Autophagy, Clinical Trials, Phase III as Topic, Combined Modality Therapy, Cytoplasmic Vesicles, Humans, Hydroxychloroquine, Immune Tolerance, Immunotherapy, Inflammation, Metabolic Networks and Pathways, Molecular Targeted Therapy, Neoplasms, Proteasome Endopeptidase Complex, Signal Transduction}, issn = {1078-0432}, doi = {10.1158/1078-0432.CCR-10-2634}, author = {Amaravadi, Ravi K and Lippincott-Schwartz, Jennifer and Yin, Xiao-Ming and Weiss, William A and Takebe, Naoko and Timmer, William and DiPaola, Robert S and Lotze, Michael T and White, Eileen} } @article {47667, title = {Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis.}, journal = {Nature methods}, volume = {8}, year = {2011}, month = {2011 Nov}, pages = {969-75}, abstract = {

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

}, keywords = {Algorithms, Membrane Proteins}, issn = {1548-7105}, doi = {10.1038/nmeth.1704}, author = {Sengupta, Prabuddha and Jovanovic-Talisman, Tijana and Skoko, Dunja and Renz, Malte and Veatch, Sarah L and Lippincott-Schwartz, Jennifer} } @article {47671, title = {A role for actin arcs in the leading-edge advance of migrating cells.}, journal = {Nature cell biology}, volume = {13}, year = {2011}, month = {2011 Apr}, pages = {371-81}, abstract = {

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.

}, keywords = {Actins, Cell Movement, Cytoskeleton, Epithelial Cells, Focal Adhesions, Myosin Type II, Pseudopodia, Recombinant Fusion Proteins}, issn = {1476-4679}, doi = {10.1038/ncb2205}, author = {Burnette, Dylan T and Manley, Suliana and Sengupta, Prabuddha and Sougrat, Rachid and Davidson, Michael W and Kachar, Bechara and Lippincott-Schwartz, Jennifer} } @article {47665, title = {Together we are stronger: fusion protects mitochondria from autophagosomal degradation.}, journal = {Autophagy}, volume = {7}, year = {2011}, month = {2011 Dec}, pages = {1568-9}, abstract = {

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.

}, keywords = {Animals, Autophagy, Cyclic AMP-Dependent Protein Kinases, Membrane Fusion, Mice, Mitochondria, Phagosomes, Signal Transduction, TOR Serine-Threonine Kinases}, issn = {1554-8635}, author = {Rambold, Angelika S and Kostelecky, Brenda and Lippincott-Schwartz, Jennifer} } @article {47669, title = {Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Jun 21}, pages = {10190-5}, abstract = {

Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by down-regulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.

}, keywords = {Animals, Autophagy, Cells, Cultured, Dynamins, Fibroblasts, GTP Phosphohydrolases, Mice, Microtubule-Associated Proteins, Mitochondria, Mitochondrial Proteins, Phagosomes, Protein Processing, Post-Translational, Starvation}, issn = {1091-6490}, doi = {10.1073/pnas.1107402108}, author = {Rambold, Angelika S and Kostelecky, Brenda and Elia, Natalie and Lippincott-Schwartz, Jennifer} } @article {47682, title = {Analysis of mitochondrial dynamics and functions using imaging approaches.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 4}, year = {2010}, month = {2010 Mar}, pages = {Unit 4.25.1-21}, abstract = {

Mitochondria are organelles that have been primarily known as the powerhouse of the cell. However, recent advances in the field have revealed that mitochondria are also involved in many other cellular activities like lipid modifications, redox balance, calcium balance, and even controlled cell death. These multifunctional organelles are motile and highly dynamic in shapes and forms; the dynamism is brought about by the mitochondria{\textquoteright}s ability to undergo fission and fusion with each other. Therefore, it is very important to be able to image mitochondrial shape changes to relate to the variety of cellular functions these organelles have to accomplish. The protocols described here will enable researchers to perform steady-state and time-lapse imaging of mitochondria in live cells by using confocal microscopy. High-resolution three-dimensional imaging of mitochondria will not only be helpful in understanding mitochondrial structure in detail but it also could be used to analyze their structural relationships with other organelles in the cell. FRAP (fluorescence recovery after photobleaching) studies can be performed to understand mitochondrial dynamics or dynamics of any mitochondrial molecule within the organelle. The microirradiation assay can be performed to study functional continuity between mitochondria. A protocol for measuring mitochondrial potential has also been included in this unit. In conclusion, the protocols described here will aid the understanding of mitochondrial structure-function relationship.

}, keywords = {Cell Survival, Electricity, Fluorescence Recovery After Photobleaching, Humans, Imaging, Three-Dimensional, Membrane Potential, Mitochondrial, Microtubules, Mitochondria, Nocodazole, Staining and Labeling, Time Factors}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0425s46}, author = {Mitra, Kasturi and Lippincott-Schwartz, Jennifer} } @article {47685, title = {Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.}, journal = {Journal of the American Chemical Society}, volume = {132}, year = {2010}, month = {2010 May 12}, pages = {6481-91}, abstract = {

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

}, keywords = {Amino Acid Sequence, Animals, Cell Survival, Cercopithecus aethiops, Color, COS Cells, Hela Cells, Humans, Light, Luminescent Proteins, Membrane Proteins, Microscopy, Molecular Imaging, Molecular Sequence Data, Mutagenesis, Photochemical Processes, Temperature}, issn = {1520-5126}, doi = {10.1021/ja100906g}, author = {Subach, Fedor V and Patterson, George H and Renz, Malte and Lippincott-Schwartz, Jennifer and Verkhusha, Vladislav V} } @article {47675, title = {Determining membrane protein topologies in single cells and high-throughput screening applications.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 5}, year = {2010}, month = {2010 Dec}, pages = {Unit 5.7}, abstract = {

Correct localization and topology are crucial for a protein{\textquoteright}s cellular function. To determine topologies of membrane proteins, a new technique, called fluorescence protease protection (FPP) assay, has recently been established. The sole requirements for FPP are the expression of fluorescent-protein fusion proteins and the selective permeabilization of the plasma membrane, permitting a wide range of cell types and organelles to be investigated. Proteins topologies in organelles like endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes have already been determined by FPP. Here, two different step-by-step protocols of the FPP assay are provided. First, we describe the FPP assay using fluorescence microscopy for single adherent cells, and second, we outline the FPP assay for high-throughput screening applications.

}, keywords = {Animals, Cell Line, Cytological Techniques, High-Throughput Screening Assays, Humans, Luminescent Proteins, Membrane Proteins, Microscopy, Fluorescence, Peptide Hydrolases, Protein Transport, Recombinant Fusion Proteins, Single-Cell Analysis}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0507s49}, author = {Wunder, Christian and Lippincott-Schwartz, Jennifer and Lorenz, Holger} } @article {47676, title = {Imaging: visualizing the possibilities.}, journal = {Journal of cell science}, volume = {123}, year = {2010}, month = {2010 Nov 1}, pages = {3619-20}, keywords = {Diagnostic Imaging, Fluorescence Recovery After Photobleaching, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Humans, Image Enhancement, Molecular Probes, Photoaffinity Labels}, issn = {1477-9137}, doi = {10.1242/jcs081539}, author = {Lippincott-Schwartz, Jennifer} } @article {47684, title = {Lipids and cholesterol as regulators of traffic in the endomembrane system.}, journal = {Annual review of biophysics}, volume = {39}, year = {2010}, month = {2010}, pages = {559-78}, abstract = {

The endomembrane system of eukaryotic cells uses membrane-enclosed carriers to move diverse macromolecules among different membrane-bound compartments, a requirement for cells to secrete and take up molecules from their environment. Two recycling pathways-biosynthetic and endocytic, each with specific lipid components-make up this system, with the Golgi apparatus mediating transport between the two. Here, we integrate lipid-based mechanisms into the description of this system. A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents. We further discuss how computational modeling can allow for interpretation of experimental findings and provide mechanistic insight into these important cellular pathways.

}, keywords = {Animals, Bacteria, Cholesterol, Endocytosis, Golgi Apparatus, Humans, Intracellular Membranes, Lipid Metabolism, Protein Transport}, issn = {1936-1238}, doi = {10.1146/annurev.biophys.093008.131357}, author = {Lippincott-Schwartz, Jennifer and Phair, Robert D} } @article {47677, title = {The long road: peering into live cells.}, journal = {Nature cell biology}, volume = {12}, year = {2010}, month = {2010 Oct}, pages = {918}, keywords = {Cell Biology, Cell Survival, Green Fluorescent Proteins, History, 20th Century, History, 21st Century, Microscopy, Fluorescence, Photobleaching, Protein Transport, Research Personnel}, issn = {1476-4679}, doi = {10.1038/ncb1010-918}, author = {Lippincott-Schwartz, Jennifer} } @article {47681, title = {Mitochondria supply membranes for autophagosome biogenesis during starvation.}, journal = {Cell}, volume = {141}, year = {2010}, month = {2010 May 14}, pages = {656-67}, abstract = {

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

}, keywords = {Animals, Cell Line, Cell Physiological Phenomena, Culture Media, Membrane Proteins, Mitochondria, Mitochondrial Membranes, Mitochondrial Proteins, Phagosomes, Rats}, issn = {1097-4172}, doi = {10.1016/j.cell.2010.04.009}, author = {Hailey, Dale W and Rambold, Angelika S and Satpute-Krishnan, Prasanna and Mitra, Kasturi and Sougrat, Rachid and Kim, Peter K and Lippincott-Schwartz, Jennifer} } @article {47686, title = {The open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death.}, journal = {Journal of virology}, volume = {84}, year = {2010}, month = {2010 Jan}, pages = {1097-109}, abstract = {

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

}, keywords = {ADP-Ribosylation Factor 1, Animals, Cell Death, Cell Membrane, Cercopithecus aethiops, Cytoplasmic Vesicles, Gene Deletion, Golgi Apparatus, Host-Pathogen Interactions, Humans, SARS Virus, Transfection, Vero Cells, Viral Structural Proteins}, issn = {1098-5514}, doi = {10.1128/JVI.01662-09}, author = {Freundt, Eric C and Yu, Li and Goldsmith, Cynthia S and Welsh, Sarah and Cheng, Aaron and Yount, Boyd and Liu, Wei and Frieman, Matthew B and Buchholz, Ursula J and Screaton, Gavin R and Lippincott-Schwartz, Jennifer and Zaki, Sherif R and Xu, Xiao-Ning and Baric, Ralph S and Subbarao, Kanta and Lenardo, Michael J} } @article {47679, title = {Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1.}, journal = {Journal of cell science}, volume = {123}, year = {2010}, month = {2010 Oct 1}, pages = {3294-302}, abstract = {

AMP-activated protein kinase (AMPK), a cellular metabolic sensor, is essential in energy regulation and metabolism. Hepatocyte polarization during liver development and regeneration parallels increased metabolism. The current study investigates the effects of AMPK and its upstream activator LKB1 on polarity and bile canalicular network formation and maintenance in collagen sandwich cultures of rat hepatocytes. Immunostaining for the apical protein ABCB1 and the tight junction marker occludin demonstrated that canalicular network formation is sequential and is associated with activation of AMPK and LKB1. AMPK and LKB1 activators accelerated canalicular network formation. Inhibition of AMPK or LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs blocked canalicular network formation. AICAR and 2-deoxyglucose, which activate AMPK, circumvented the inhibitory effect of kinase-dead LKB1 on canalicular formation, indicating that AMPK directly affects canalicular network formation. After the canalicular network was formed, inhibition of AMPK and LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs resulted in loss of canalicular network, indicating that AMPK and LKB1 also participate in network maintenance. In addition, activation of AMPK and LKB1 prevented low-Ca(2+)-mediated disruption of the canalicular network and tight junctions. These studies reveal that AMPK and its upstream kinase, LKB1, regulate canalicular network formation and maintenance.

}, keywords = {Animals, Bile Canaliculi, Cell Polarity, Cells, Cultured, Cloning, Molecular, Enzyme Activation, Hepatocytes, Male, Mutant Proteins, Organ Culture Techniques, Organogenesis, Protein Kinases, Protein-Serine-Threonine Kinases, Rats, Rats, Sprague-Dawley}, issn = {1477-9137}, doi = {10.1242/jcs.068098}, author = {Fu, Dong and Wakabayashi, Yoshiyuki and Ido, Yasuo and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {47680, title = {Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics.}, journal = {Methods in enzymology}, volume = {475}, year = {2010}, month = {2010}, pages = {109-20}, abstract = {

Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues. Here we describe the method of single-molecule tracking photoactivated localization microscopy (sptPALM) and discuss how its use can contribute to a quantitative understanding of fundamental cellular processes.

}, keywords = {Fluorescent Dyes, Light, Membrane Proteins, Microscopy, Molecular Dynamics Simulation, Molecular Motor Proteins}, issn = {1557-7988}, doi = {10.1016/S0076-6879(10)75005-9}, author = {Manley, Suliana and Gillette, Jennifer M and Lippincott-Schwartz, Jennifer} } @article {47678, title = {Starved cells use mitochondria for autophagosome biogenesis.}, journal = {Cell cycle (Georgetown, Tex.)}, volume = {9}, year = {2010}, month = {2010 Sep 15}, pages = {3633-4}, keywords = {Autophagy, Cytochromes b5, Endoplasmic Reticulum, Microtubule-Associated Proteins, Mitochondria, Phosphatidylethanolamines, Phosphatidylserines}, issn = {1551-4005}, doi = {10.4161/cc.9.18.13170}, author = {Rambold, Angelika S and Lippincott-Schwartz, Jennifer} } @article {47683, title = {Superresolution imaging using single-molecule localization.}, journal = {Annual review of physical chemistry}, volume = {61}, year = {2010}, month = {2010}, pages = {345-67}, abstract = {

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (approximately 10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.

}, keywords = {Image Interpretation, Computer-Assisted, Luminescent Proteins, Microscopy, Fluorescence, Microscopy, Polarization}, issn = {1545-1593}, doi = {10.1146/annurev.physchem.012809.103444}, author = {Patterson, George and Davidson, Michael and Manley, Suliana and Lippincott-Schwartz, Jennifer} } @article {927, title = {Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {106}, year = {2009}, month = {2009 Mar 3}, pages = {3125-30}, abstract = {

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

}, keywords = {Animals, Cell Line, Cercopithecus aethiops, Fluorescence, Humans, Imaging, Interference, Microscopy, Microtubules, Three-Dimensional}, issn = {1091-6490}, doi = {10.1073/pnas.0813131106}, author = {Shtengel, Gleb and Galbraith, James A and Galbraith, Catherine G and Lippincott-Schwartz, Jennifer and Gillette, Jennifer M and Manley, Suliana and Sougrat, Rachid and Waterman, Clare M and Kanchanawong, Pakorn and Davidson, Michael W and Fetter, Richard D and Hess, Harald F} } @article {2080, title = {High-density mapping of single-molecule trajectories with photoactivated localization microscopy. (With commentary)}, journal = {Nature Methods}, volume = {5}, year = {2008}, month = {2008 Feb}, pages = {155-7}, abstract = {

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

Commentary: As a stepping stone to true live cell PALM (see above), our collaborator Jennifer Lippincott-Schwartz suggested using the sparse photoactivation principle of PALM to track the nanoscale motion of thousands of individual molecules within a single living cell. Termed single particle tracking PALM (sptPALM), Jennifer{\textquoteright}s postdocs Suliana Manley and Jen Gillette used the method in our PALM rig to create spatially resolved maps of diffusion rates in the plasma membrane of live cells. sptPALM is a powerful tool to study the active cytoskeletal or passive diffusional transport of individual molecules with far more measurements per cell than is possible without sparse photoactivation.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Fluorescence, Image Enhancement, Membrane Proteins, Microscopy}, issn = {1548-7105}, doi = {10.1038/nmeth.1176}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18193054}, author = {Manley, Suliana and Gillette, Jennifer M and Patterson, George H and Shroff, Hari and Hess, Harald F and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {2083, title = {Imaging intracellular fluorescent proteins at nanometer resolution. (With commentary)}, journal = {Science}, volume = {313}, year = {2006}, month = {2006 Sep 15}, pages = {1642-5}, abstract = {

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method{\textendash}termed photoactivated localization microscopy{\textendash}to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Commentary: The original PALM paper by myself and my friend and co-inventor Harald Hess, spanning the before- and after-HHMI eras. Submitted and publicly presented months before other publications in the same year, the lessons of the paper remain widely misunderstood: 1) localization precision is not resolution; 2) the ability to resolve a few molecules by the Rayleigh criterion in a diffraction limited region (DLR) does not imply the ability to resolve structures of arbitrary complexity at the same scale; 3) true resolution well beyond the Abbe limit requires the ability to isolate and localize hundreds or thousands of molecules in one DLR; and 4) certain photoactivatable fluorescent proteins (PA-FPs) and caged dyes can be isolated and precisely localized at such densities; yielding true resolution down to \ 20 nm. The molecular densities we demonstrate (105 molecules/m2) are more than two orders of magnitude greater than in later papers that year (implying ten-fold better true resolution) {\textendash} indeed, these papers demonstrate densities only comparable to earlier spectral or photobleaching based isolation methods. We validate our claims by correlative electron microscopy, and demonstrate the outstanding advantages of PA-FPs for superresolution microscopy: minimally perturbative sample preparation; high labeling densities; close binding to molecular targets; and zero non-specific background.

}, keywords = {Actins, Algorithms, Animals, Cell Line, Cell Membrane, Cercopithecus aethiops, COS Cells, Fluorescence, Focal Adhesions, gag, Gene Products, HIV-1, Light, Luminescent Proteins, Lysosomes, Microscopy, Mitochondria, Nanotechnology, Organelles, Photobleaching, Proteins, Pseudopodia, Recombinant Fusion Proteins, Vinculin}, issn = {1095-9203}, doi = {10.1126/science.1127344}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16902090}, author = {Betzig, Eric and Patterson, George H and Sougrat, Rachid and Lindwasser, O Wolf and Olenych, Scott and Bonifacino, Juan S and Davidson, Michael W and Lippincott-Schwartz, Jennifer and Hess, Harald F} }