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11 Janelia Publications

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    11/05/15 | Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline.
    Xie J, Wooten M, Tran V, Chen B, Pozmanter C, Simbolon C, Betzig E, Chen X
    Cell. 2015 Nov 5;163(4):920-33. doi: 10.1016/j.cell.2015.10.002

    A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

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    11/01/15 | Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.
    Thievessen I, Fakhri N, Steinwachs J, Kraus V, McIsaac RS, Gao L, Chen B, Baird MA, Davidson MW, Betzig E, Oldenbourg R, Waterman CM, Fabry B
    FASEB Journal. 2015 Nov;29(11):4555-67. doi: 10.1096/fj.14-268235

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.-Thievessen, I., Fakhri, N., Steinwachs, J., Kraus, V., McIsaac, R. S., Gao, L., Chen, B.-C., Baird, M. A., Davidson, M. W., Betzig, E., Oldenbourg, R., Waterman, C., M., Fabry, B. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

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    10/01/15 | Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus.
    Yamashita N, Morita M, Legant WR, Chen B, Betzig E, Yokota H, Mimori-Kiyosue Y
    Journal of Biomedical Optics. 2015 Oct 1;20(10):101206. doi: 10.1117/1.JBO.20.10.101206
    08/28/15 | Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.
    Li D, Shao L, Chen B, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E
    Science (New York, N.Y.). 2015 Aug 28;349(6251):. doi: 10.1126/science.aab3500

    Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

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    06/18/15 | Single molecules, cells, and super-resolution optics (Nobel Lecture).
    Betzig E
    Angewandte Chemie (International Edition in English). 2015 Jun 18:. doi: 10.1002/anie.201501003

    The resolution of a microscope is determined by the diffraction limit in classical microscopy, whereby objects that are separated by half a wavelength can no longer be visually separated. To go below the diffraction limit required several tricks and discoveries. In his Nobel Lecture, E. Betzig describes the developments that have led to modern super high-resolution microscopy.

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    06/15/15 | Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.
    Wang K, Sun W, Richie CT, Harvey BK, Betzig E, Ji N
    Nature Communications. 2015-Jun-15;6:7276. doi: 10.1038/ncomms8276

    Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

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    06/01/15 | High-resolution live imaging reveals axon-glia interactions during peripheral nerve injury and repair in zebrafish.
    Xiao Y, Faucherre A, Pola-Morell L, Heddleston JM, Liu T, Chew T, Sato F, Sehara-Fujisawa A, Kawakami K, López-Schier H
    Disease Models & Mechanisms. 2015 Jun 1;8(6):553-64. doi: 10.1242/dmm.018184

    Neural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of Büngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation.

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    05/21/15 | Imaging live-cell dynamics and structure at the single-molecule level.
    Liu Z, Lavis LD, Betzig E
    Molecular Cell. 2015 May 21;58(4):644-59. doi: 10.1016/j.molcel.2015.02.033

    Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen.

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    05/19/15 | Actin depletion initiates events leading to granule secretion at the immunological synapse.
    Ritter AT, Asano Y, Stinchcombe JC, Dieckmann NM, Chen B, Gawden-Bone C, van Engelenburg S, Legant W, Gao L, Davidson MW, Betzig E, Lippincott-Schwartz J, Griffiths GM
    Immunity. 2015 May 19;42(5):864-76. doi: 10.1016/j.immuni.2015.04.013

    Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

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    04/07/15 | Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edge of motile cells and on protrusions of immobile cells.
    Kural C, Akatay AA, Gaudin R, Chen B, Legant WR, Betzig E, Kirchhausen T
    Molecular Biology of the Cell. 2015 Apr 7;26(11):2044-53. doi: 10.1091/mbc.E15-01-0055

    Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In experiments reported here, we have used spinning disk confocal and lattice light sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2-EGFP and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium, as well as at the approximate boundary between the lamellipodium and lamella, and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary-coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.

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