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66 Janelia Publications

Showing 11-20 of 66 results
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    05/24/21 | A general method to improve fluorophores using deuterated auxochromes.
    Grimm JB, Xie L, Casler JC, Patel R, Tkachuk AN, Falco N, Choi H, Lippincott-Schwartz J, Brown TA, Glick BS, Liu Z, Lavis LD
    JACS Au. 2021 May 24;1(5):690-6. doi: 10.1021/jacsau.1c00006

    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—thereby increasing the brightness and photostability of fluorophores—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.

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    05/08/21 | Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome
    Govind AP, Jeyifous O, Russell TA, Yi Z, Weigel AV, Ramaprasad A, Newell L, Ramos W, Valbuena FM, Casler JC, Yan J, Glick BS, Swanson GT, Lippincott-Schwartz J, Green WN
    bioRxiv. 05/2021:. doi: 10.1101/2021.04.06.438745

    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’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.

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    05/01/21 | RNA transport and local translation in neurodevelopmental and neurodegenerative disease.
    Fernandopulle MS, Lippincott-Schwartz J, Ward ME
    Nature Neuroscience. 2021 May 01;24(5):622-32. doi: 10.1038/s41593-020-00785-2

    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's role in RNA localization and illuminating their unique contributions to neurodegeneration.

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    04/29/21 | ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.
    Weigel AV, Chang C, Shtengel G, Xu CS, Hoffman DP, Freeman M, Iyer N, Aaron J, Khuon S, Bogovic J, Qiu W, Hess HF, Lippincott-Schwartz J
    Cell. 2021 Apr 29;184(9):2412. doi: 10.1016/j.cell.2021.03.035

    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.

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    03/03/21 | Actin cables and comet tails organize mitochondrial networks in mitosis.
    Moore AS, Coscia SM, Simpson CL, Ortega FE, Wait EC, Heddleston JM, Nirschl JJ, Obara CJ, Guedes-Dias P, Boecker CA, Chew T, Theriot JA, Lippincott-Schwartz J, Holzbaur EL
    Nature. 2021 Mar 03;591(7851):659-664. doi: 10.1038/s41586-021-03309-5

    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.

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    02/10/21 | Biomolecular Condensates and Their Links to Cancer Progression.
    Cai D, Liu Z, Lippincott-Schwartz J
    Trends in Biochemical Sciences. 2021 Feb 10:. doi: 10.1016/j.tibs.2021.01.002

    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.

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    02/01/21 | Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.
    Kanfer G, Sarraf SA, Maman Y, Baldwin H, Dominguez-Martin E, Johnson KR, Ward ME, Kampmann M, Lippincott-Schwartz J, Youle RJ
    Journal of Cell Biology. 2021 Feb 01;220(2):. doi: 10.1083/jcb.202006180

    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.

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    12/15/20 | In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning.
    Gur D, Bain EJ, Johnson KR, Aman AJ, Pasoili HA, Flynn JD, Allen MC, Deheyn DD, Lee JC, Lippincott-Schwartz J, Parichy DM
    Nature Communications. 2020 Dec 15;11(1):6391. doi: 10.1038/s41467-020-20088-1

    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'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.

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    12/01/20 | The evolution of a cell biologist.
    Lippincott-Schwartz J
    Molecular Biology of the Cell. 2020 Dec 01;31(25):2763-2767. doi: 10.1091/mbc.E20-09-0603

    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.

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    11/01/20 | 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 Nov 01;93:79-94. 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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