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155 Publications

Showing 81-90 of 155 results
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    10/28/16 | Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.
    Nixon-Abell J, Obara CJ, Weigel AV, Li D, Legant WR, Xu C, Pasolli HA, Harvey K, Hess HF, Betzig E, Blackstone C, Lippincott-Schwartz J
    Science (New York, N.Y.). 2016 Oct 28;354(6311):433-46. doi: 10.1126/science.aaf3928

    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 “sheet” proliferation after overexpression of tubular junction–forming proteins.

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    10/24/16 | Bright photoactivatable fluorophores for single-molecule imaging.
    Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
    Nature Methods. 2016 Oct 24;13(12):985-8. doi: 10.1038/nmeth.4034

    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.

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    08/01/16 | Midbody remnant licenses primary cilia formation in epithelial cells.
    Ott CM
    The Journal of Cell Biology. 2016 Aug 1;214(3):237-9. doi: 10.1083/jcb.201607046

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model.

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    02/06/16 | Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo.
    Rikhy R, Mavrakis M, Lippincott-Schwartz J
    Biology open. 2015;4(3):301-11. doi: 10.1242/bio.20149936

    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.

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    02/03/16 | Intracellular and extracellular forces drive primary cilia movement.
    Battle C, Ott CM, Burnette DT, Lippincott-Schwartz J, Schmidt CF
    Proceedings of the National Academy of Sciences of the United States of America. 2015 Feb 3;112(5):1410-5. doi: 10.1073/pnas.1421845112

    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.

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    03/03/15 | Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry.
    Lippincott-Schwartz J
    Proceedings of the National Academy of Sciences of the United States of America. 2015 Mar 3;112(9):2630-2. doi: 10.1073/pnas.1500784112
    02/16/15 | Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.
    Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang Y, Daar IO, Lopes S, Lippincott-Schwartz J, Jackson PK, Caplan S, Westlake CJ
    Nature Cell Biology. 2015 Feb 16;17(4):228-40. doi: 10.1038/ncb3109

    Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–​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–​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.

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    02/05/15 | Deacetylation of nuclear LC3 drives autophagy initiation under starvation.
    Huang R, Xu Y, Wan W, Shou X, Qian J, You Z, Liu B, Chang C, Zhou T, Lippincott-Schwartz J, Liu W
    Molecular cell. 2015 Feb 5;57(3):456-66. doi: 10.1016/j.molcel.2014.12.013

    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'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.

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    01/01/15 | Photocontrollable fluorescent proteins for superresolution imaging.
    Shcherbakova DM, Sengupta P, Lippincott-Schwartz J, Verkhusha VV
    Annual review of biophysics. 2014;43:303-29. doi: 10.1146/annurev-biophys-051013-022836

    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.

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    12/19/14 | Cell Biology. Fixing problems with cell lines.
    Lorsch JR, Collins FS, Lippincott-Schwartz J
    Science (New York, N.Y.). 2014 Dec 19;346(6216):1452-3. doi: 10.1126/science.1259110