Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

filters_region_cap | custom

Filter

facetapi-Q2b17qCsTdECvJIqZJgYMaGsr8vANl1n | block
facetapi-W9JlIB1X0bjs93n1Alu3wHJQTTgDCBGe | block
facetapi-61yz1V0li8B1bixrCWxdAe2aYiEXdhd0 | block
facetapi-PV5lg7xuz68EAY8eakJzrcmwtdGEnxR0 | block
general_search_page-panel_pane_1 | views_panes

94 Janelia Publications

Showing 31-40 of 94 results
Your Criteria:
    01/23/17 | Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.
    Beach JR, Bruun KS, Shao L, Li D, Swider Z, Remmert K, Zhang Y, Conti MA, Adelstein RS, Rusan NM, Betzig E, Hammer JA
    Nature Cell Biology. 2017 Jan 23;19(2):85-93. doi: 10.1038/ncb3463

    The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

    View Publication Page
    12/13/16 | A plasma membrane template for macropinocytic cups.
    Veltman DM, Williams TD, Bloomfield G, Chen B, Betzig E, Insall RH, Kay RR
    eLife. 2016 Dec 13;5:. doi: 10.7554/eLife.20085

    Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.

    View Publication Page
    11/14/16 | Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells.
    Hayer A, Shao L, Chung M, Joubert L, Yang HW, Tsai F, Bisaria A, Betzig E, Meyer T
    Nature Cell Biology. 2016 Nov 14;18(12):1311-23. doi: 10.1038/ncb3438

    The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, ‘cadherin fingers’, which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.

    View Publication Page
    10/31/16 | Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.
    Murugesan S, Hong J, Yi J, Li D, Beach JR, Shao L, Meinhardt J, Madison G, Wu X, Betzig E, Hammer JA
    The Journal of Cell Biology. 2016 Oct 31;215(3):383-99. doi: 10.1083/jcb.201603080

    Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

    View Publication Page
    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.

    View Publication Page
    10/25/16 | V-1 regulates capping protein activity in vivo.
    Jung G, Alexander CJ, Wu XS, Piszczek G, Chen B, Betzig E, Hammer JA
    Proceedings of the National Academy of Sciences of the United States of America. 2016 Oct 25;113(43):E6610-9. doi: 10.1073/pnas.1605350113

    Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

    View Publication Page
    09/20/16 | Bessel beam plane illumination microscope.
    Betzig E
    USPTO. 2016 Sep 20;B2:

    A microscope has a light source for generating a light beam having a wavelength, λ, and beam-forming optics configured for receiving the light beam and generating a Bessel-like beam that is directed into a sample. The beam-forming optics include an excitation objective having an axis oriented in a first direction. Imaging optics are configured for receiving light from a position within the sample that is illuminated by the Bessel-like beam and for imaging the received light on a detector. The imaging optics include a detection objective having an axis oriented in a second direction that is non-parallel to the first direction. A detector is configured for detecting signal light received by the imaging optics, and an aperture mask is positioned.

    View Publication Page
    08/25/16 | Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy.
    Zhang X, Zhang M, Li D, He W, Peng J, Betzig E, Xu P
    Proceedings of the National Academy of Sciences of the United States of America. 2016 Aug 25;113(37):10364-9. doi: 10.1073/pnas.1611038113

    Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

    View Publication Page
    08/17/16 | Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy.
    Aguet F, Upadhyayula S, Gaudin R, Chou Y, Cocucci E, He K, Chen B, Mosaliganti K, Pasham M, Skillern W, Legant WR, Liu T, Findlay G, Marino E, Danuser G, Megason S, Betzig E, Kirchhausen T
    Molecular Biology of the Cell. 2016 Aug 17;27(22):3418-35. doi: 10.1091/mbc.E16-03-0164

    Membrane remodeling is an essential part for transfer of components to and from the cell surface and membrane-bound organelles, and for changes in cell shape, particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging, mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and non-invasive illumination of the newly developed lattice light sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and showed that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and remained relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and in multicellular assemblies.

    View Publication Page
    08/03/16 | Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.
    Li L, Liu H, Dong P, Li D, Legant WR, Grimm JB, Lavis LD, Betzig E, Tjian R, Liu Z
    eLife. 2016 Aug 03;5:. doi: 10.7554/eLife.17056

    The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

    View Publication Page