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21 Janelia Publications

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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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    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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    09/25/17 | Cell volume change through water efflux impacts cell stiffness and stem cell fate.
    Guo M, Pegoraro AF, Mao A, Zhou EH, Arany PR, Han Y, Burnette DT, Jensen MH, Kasza KE, Moore JR, Mackintosh FC, Fredberg JJ, Mooney DJ, Lippincott-Schwartz J, Weitz DA
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Sep 25;114(41):E8618-27. doi: 10.1073/pnas.1705179114

    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.

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    07/17/17 | A consensus view of ESCRT-mediated Human Immunodeficiency Virus Type 1 abscission.
    Lippincott-Schwartz J, Freed EO, van Engelenburg SB
    Annual Review of Virology. 2017 Jul 17;4(1):309-25. doi: 10.1146/annurev-virology-101416-041840

    The strong dependence of retroviruses, such as human immunodeficiency virus type 1 (HIV-1), on host cell factors is no more apparent than when the endosomal sorting complex required for transport (ESCRT) machinery is purposely disengaged. The resulting potent inhibition of retrovirus release underscores the importance of understanding fundamental structure-function relationships at the ESCRT-HIV-1 interface. Recent studies utilizing advanced imaging technologies have helped clarify these relationships, overcoming hurdles to provide a range of potential models for ESCRT-mediated virus abscission. Here, we discuss these models in the context of prior work detailing ESCRT machinery and the HIV-1 release process. To provide a template for further refinement, we propose a new working model for ESCRT-mediated HIV-1 release that reconciles disparate and seemingly conflicting studies. Expected final online publication date for the Annual Review of Virology Volume 4 is September 29, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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    06/29/17 | Rational engineering of photoconvertible fluorescent proteins for dual-color fluorescence nanoscopy enabled by a triplet-state mechanism of primed conversion.
    Mohr MA, Kobitski AY, Sabater LR, Nienhaus K, Obara CJ, Lippincott-Schwartz J, Nienhaus GU, Pantazis P
    Angewandte Chemie (International ed. in English). 2017 Jun 29:. doi: 10.1002/anie.201706121

    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.

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    05/24/17 | Applying systems-level spectral imaging and analysis to reveal the organelle interactome.
    Valm AM, Cohen S, Legant WR, Melunis J, Hershberg U, Wait E, Cohen AR, Davidson MW, Betzig E, Lippincott-Schwartz J
    Nature. 2017 May 24:. doi: 10.1038/nature22369

    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.

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