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72 Publications

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    08/08/17 | Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.
    Ritter AT, Kapnick SM, Murugesan S, Schwartzberg PL, Griffiths GM, Lippincott-Schwartz J
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Aug 08;114(32):E6585-94. doi: 10.1073/pnas.1710751114

    CD8(+) cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.

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    03/01/17 | Myosin VI facilitates connexin 43 gap junction accretion.
    Waxse BJ, Sengupta P, Hesketh GG, Lippincott-Schwartz J, Buss F
    Journal of Cell Science. 2017 03 01;130(5):827-840. doi: 10.1242/jcs.199083

    In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI.

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    12/08/16 | Sonic Hedgehog Pathway activation increases mitochondrial abundance and activity in hippocampal neurons.
    Yao PJ, Manor U, Petralia RS, Brose RD, Wu RT, Ott C, Wang Y, Charnoff A, Lippincott-Schwartz J, Mattson MP
    Molecular Biology of the Cell. 2016 Dec 08:. doi: 10.1091/mbc.E16-07-0553

    Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. In this study we present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway impacts multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively, our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

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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
    12/15/14 | Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
    Xia P, Liu X, Wu B, Zhang S, Song X, Yao PY, Lippincott-Schwartz J, Yao X
    Molecular biology of the cell. 2014 Dec 15;25(25):4166-73. doi: 10.1091/mbc.E14-06-1133

    Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure-function relationship of EB1 by generating EB1-PACF dimers (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.

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    11/08/14 | Photohighlighting approaches to access membrane dynamics of the Golgi apparatus.
    Sengupta P, Lippincott-Schwartz J
    Methods in cell biology. 2013;118:217-34. doi: 10.1016/B978-0-12-417164-0.00013-6

    By providing quantitative, visual data of live cells, fluorescent protein-based microscopy techniques are furnishing novel insights into the complexities of membrane trafficking pathways and organelle dynamics. In this chapter, we describe experimental protocols employing fluorescent protein-based photohighlighting techniques to quantify protein movement into and out of the Golgi apparatus, an organelle that serves as the central sorting and processing station of the secretory pathway. The methods allow kinetic characteristics of Golgi-associated protein trafficking to be deciphered, which can help clarify how the Golgi maintains itself as a steady-state structure despite a continuous flux of secretory cargo passing into and out of this organelle. The guidelines presented in this chapter can also be applied to examine the dynamics of other intracellular organelle systems, elucidating mechanisms for how proteins are maintained in specific organelles and/or circulated to other destinations within the cell.

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