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66 Janelia Publications

Showing 41-50 of 66 results
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    08/17/18 | mTOR-dependent phosphorylation controls TFEB nuclear export.
    Napolitano G, Esposito A, Choi H, Matarese M, Benedetti V, Di Malta C, Monfregola J, Medina DL, Lippincott-Schwartz J, Ballabio A
    Nature Communications. 2018 Aug 17;9(1):3312. doi: 10.1038/s41467-018-05862-6

    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.

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    08/17/18 | The development and enhancement of FRAP as a key tool for investigating protein dynamics.
    Lippincott-Schwartz J, Snapp EL, Phair RD
    Biophysical Journal. 2018 Aug 17;115(7):1146-55. doi: 10.1016/j.bpj.2018.08.007

    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'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.

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    08/13/18 | Triggered cell-cell fusion assay for cytoplasmic and organelle intermixing studies.
    Feliciano D, Nixon-Abell J, Lippincott-Schwartz J
    Current Protocols in Cell Biology. 2018 Aug 13;81(1):e61. doi: 10.1002/cpcb.61

    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. © 2018 by John Wiley & Sons, Inc.

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    08/01/18 | Interacting organelles.
    Cohen S, Valm AM, Lippincott-Schwartz J
    Current Opinion in Cell Biology. 2018 Aug;53:84-91. doi: 10.1016/j.ceb.2018.06.003

    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.

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    06/01/18 | Monitoring the effects of pharmacological reagents on mitochondrial morphology.
    Fu D, Lippincott-Schwartz J
    Current Protocols in Cell Biology. 2018 Jun;79(1):e45. doi: 10.1002/cpcb.45

    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. © 2018 by John Wiley & Sons, Inc.

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    06/01/18 | Multispectral live-cell imaging.
    Cohen S, Valm AM, Lippincott-Schwartz J
    Current Protocols in Cell Biology. 2018 Jun;79(1):e46. doi: 10.1002/cpcb.46

    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. © 2018 by John Wiley & Sons, Inc.

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    02/20/18 | VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.
    Ott C, Nachmias D, Adar S, Jarnik M, Sherman S, Birnbaum RY, Lippincott-Schwartz J, Elia N
    Scientific Reports. 2018 Feb 20;8(1):3353. doi: 10.1038/s41598-018-21491-x

    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'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'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.

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    02/18/18 | Transport and sorting in the Golgi complex: multiple mechanisms sort diverse cargo.
    Boncampain G, Weigel AV
    Current Opinion in Cell Biology. 2018 Feb ;50:. doi: 10.1016/j.ceb.2018.03.002

    At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport.

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    11/07/17 | Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.
    Pak AJ, Grime JM, Sengupta P, Chen AK, Durumeric AE, Srivastava A, Yeager M, Briggs JA, Lippincott-Schwartz J, Voth GA
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Nov 07;114(47):E10056-65. doi: 10.1073/pnas.1706600114

    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.

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    10/31/17 | Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.
    Stefan CJ, Trimble WS, Grinstein S, Drin G, Reinisch K, De Camilli P, Cohen S, Valm AM, Lippincott-Schwartz J, Levine TP, Iaea DB, Maxfield FR, Futter CE, Eden ER, Judith D, van Vliet AR, Agostinis P, Tooze SA, Sugiura A, McBride HM
    BMC Biology. 2017 Oct 31;15(1):102. doi: 10.1186/s12915-017-0432-0

    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.

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