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47 Janelia Publications

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    07/27/20 | A general method to optimize and functionalize red-shifted rhodamine dyes.
    Grimm JB, Tkachuk AN, Xie L, Choi H, Mohar B, Falco N, Schaefer K, Patel R, Zheng Q, Liu Z, Lippincott-Schwartz J, Brown TA, Lavis LD
    Nature Methods. 2020 Jul 27:. doi: 10.1038/s41592-020-0909-6

    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 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

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    07/10/20 | Revisiting Membrane Microdomains and Phase Separation: A Viral Perspective
    Sengupta P, Lippincott-Schwartz J
    Viruses. 2020 Jul 10;12(7):745. doi: 10.3390/v12070745

    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.

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    06/17/20 | Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking
    Omari S, Makareeva E, Gorrell L, Jarnik M, Lippincott-Schwartz J, Leikin S
    Matrix Biology. 2020 Jun 17:. doi: 10.1016/j.matbio.2020.06.002

    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.

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    03/31/20 | ER membranes exhibit phase behavior at sites of organelle contact.
    King C, Sengupta P, Seo AY, Lippincott-Schwartz J
    Proceedings of the National Academy of Sciences of the United States of America. 2020 March 31;117(13):7225-7235. doi: 10.1073/pnas.1910854117

    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–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.

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    02/26/20 | Nicotine exposure and neuronal activity regulate Golgi membrane dispersal and distribution
    Govind AP, Jeyifous O, Russell TA, Vaasjo LO, Yi Z, Weigel AV, Newell L, Koranda JL, Singh K, Valbuena F, Glick BS, Mukherjee J, Lippincott-Schwartz J, Zhuang X, Green WN
    bioRxiv. 2020 Feb 26:

    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’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 “ministacks”, 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.

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    01/17/20 | Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
    Hoffman DP, Shtengel G, Xu CS, Campbell KR, Freeman M, Wang L, Milkie DE, Pasolli HA, Iyer N, Bogovic JA, Stabley DR, Shirinifard A, Pang S, Peale D, Schaefer K, Pomp W, Chang C, Lippincott-Schwartz J, Kirchhausen T, Solecki DJ, Betzig E, Hess HF
    Science. 2020 Jan 17;367(6475):. doi: 10.1126/science.aaz5357

    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.

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    12/18/19 | Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression.
    Cai D, Feliciano D, Dong P, Flores E, Gruebele M, Porat-Shliom N, Sukenik S, Liu Z, Lippincott-Schwartz J
    Nature Cell Biology. 2019 Dec;21(12):1578-1589. doi: 10.1038/s41556-019-0433-z

    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.

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    09/25/19 | Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.
    Zheng Q, Ayala AX, Chung I, Weigel AV, Ranjan A, Falco N, Grimm JB, Tkachuk AN, Wu C, Lippincott-Schwartz J, Singer RH, Lavis LD
    ACS Central Science. 2019 Sep 25;5(9):1602-1613. doi: 10.1021/acscentsci.9b00676

    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.

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    09/19/19 | RNA granules hitchhike on lysosomes for long-distance transport, Using annexin A11 as a molecular tether.
    Liao Y, Fernandopulle MS, Wang G, Choi H, Hao L, Drerup CM, Patel R, Qamar S, Nixon-Abell J, Shen Y, Meadows W, Vendruscolo M, Knowles TP, Nelson M, Czekalska MA, Musteikyte G, Gachechiladze MA, Stephens CA, Pasolli HA, Forrest LR, St George-Hyslop P, Lippincott-Schwartz J, Ward ME
    Cell. 2019 Sep 19;179(1):147-164.e20. doi: 10.1016/j.cell.2019.08.050

    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.

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    09/01/19 | A neuron-glia Co-culture system for studying intercellular lipid transport.
    Ioannou MS, Liu Z, Lippincott-Schwartz J
    Curr Protoc Cell Biol. 2019 Sep 01;84(1):e95. doi: 10.1002/cpcb.95

    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. © 2019 The Authors.

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