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106 Publications

Showing 41-50 of 106 results
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    06/10/16 | in vivo brain imaging with adaptive optical microscope.
    Wang K, Sun W, Ji N, Betzig E
    Conference on Lasers and Electro-Optics (CLEO): Applications and Technology. 2016 Jun :AM40.1. doi: 10.1364/CLEO_AT.2016.AM4O.1

    The diffraction limited resolution of two photon and confocal microscope can be recovered using adaptive optics to explore the detailed neuronal network in the brains of zebrafish and mouse in vivo.

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    04/14/16 | Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?
    Imhof S, Fragoso C, Hemphill A, von Schubert C, Li D, Legant W, Betzig E
    F1000 Research. 2016 Apr 14;5:682. doi: 10.12688/f1000research.8249.1

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

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    03/07/16 | High-density three-dimensional localization microscopy across large volumes.
    Legant WR, Shao L, Grimm JB, Brown TA, Milkie DE, Avants BB, Lavis LD, Betzig E
    Nature Methods. 2016 Mar 7:. doi: 10.1038/nmeth.3797

    Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

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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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