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92 Janelia Publications

Showing 61-70 of 92 results
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    10/29/13 | Fast structural responses of gap junction membrane domains to AB5 toxins.
    Majoul IV, Gao L, Betzig E, Onichtchouk D, Butkevich E, Kozlov Y, Bukauskas F, Bennett MV, Lippincott-Schwartz J, Duden R
    Proceedings of the National Academy of Sciences of the United States of America. 2013 Oct 29;110(44):E4125-33. doi: 10.1073/pnas.1315850110

    Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast ( 500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

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    04/24/13 | Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes.
    Grimm JB, Sung AJ, Legant WR, Hulamm P, Matlosz SM, Betzig E, Lavis LD
    ACS Chemical Biology. 2013 Apr 24;8(6):1303-10. doi: 10.1021/cb4000822

    Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions of the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent and fluorogenic analogues of the classic fluorescein and rhodamine 110 fluorophores by replacement of the xanthene oxygen with a quaternary carbon. These anthracenyl "carbofluorescein" and "carborhodamine 110" fluorophores exhibit excellent fluorescent properties and can be masked with enzyme- and photolabile groups to prepare high-contrast fluorogenic molecules useful for live cell imaging experiments and super-resolution microscopy. Our divergent approach to these red-shifted dye scaffolds will enable the preparation of numerous novel fluorogenic probes with high biological utility.

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    03/22/13 | A localized Wnt signal orients asymmetric stem cell division in vitro.
    Habib SJ, Chen B, Tsai F, Anastassiadis K, Meyer T, Betzig E, Nusse R
    Science. 2013 Mar 22;339(6126):1445-8. doi: 10.1126/science.1231077

    Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

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    01/15/13 | Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.
    Legant WR, Choi CK, Miller JS, Shao L, Gao L, Betzig E, Chen CS
    Proceedings of the National Academy of Sciences of the United States of America. 2013 Jan 15;110(3):881-6. doi: 10.1073/pnas.1207997110

    Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

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    12/07/12 | Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.
    Gao L, Shao L, Higgins CD, Poulton JS, Peifer M, Davidson MW, Wu X, Goldstein B, Betzig E
    Cell. 2012 Dec 7;151(6):1370-85. doi: 10.1016/j.cell.2012.10.008

    Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

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    11/01/12 | Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.
    Giurumescu CA, Kang S, Planchon TA, Betzig E, Bloomekatz J, Yelon D, Cosman P, Chisholm AD
    Development. 2012 Nov;139(22):4271-9. doi: 10.1242/dev.086256

    A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to  350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

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    06/01/12 | Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras.
    Shao L, Winoto L, Agard DA, Gustafsson MG, Sedat JW
    Journal of Microscopy. 2012 Jun;246:229-36. doi: 10.1111/j.1365-2818.2012.03604.x

    In an interferometer-based fluorescence microscope, a beam splitter is often used to combine two emission wavefronts interferometrically. There are two perpendicular paths along which the interference fringes can propagate and normally only one is used for imaging. However, the other path also contains useful information. Here we introduced a second camera to our interferometer-based three-dimensional structured-illumination microscope (I(5)S) to capture the fringes along the normally unused path, which are out of phase by π relative to the fringes along the other path. Based on this complementary phase relationship and the well-defined phase interrelationships among the I(5)S data components, we can deduce and then computationally eliminate the path length errors within the interferometer loop using the simultaneously recorded fringes along the two imaging paths. This self-correction capability can greatly relax the requirement for eliminating the path length differences before and maintaining that status during each imaging session, which are practically challenging tasks. Experimental data is shown to support the theory.

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    03/09/12 | Triggering a cell shape change by exploiting preexisting actomyosin contractions.
    Roh-Johnson M, Shemer G, Higgins CD, McClellan JH, Werts AD, Tulu US, Gao L, Betzig E, Kiehart DP, Goldstein B
    Science. 2012 Mar 9;335(6073):1232-5. doi: 10.1126/science.1217869

    Apical constriction changes cell shapes, driving critical morphogenetic events, including gastrulation in diverse organisms and neural tube closure in vertebrates. Apical constriction is thought to be triggered by contraction of apical actomyosin networks. We found that apical actomyosin contractions began before cell shape changes in both Caenorhabitis elegans and Drosophila. In C. elegans, actomyosin networks were initially dynamic, contracting and generating cortical tension without substantial shrinking of apical surfaces. Apical cell-cell contact zones and actomyosin only later moved increasingly in concert, with no detectable change in actomyosin dynamics or cortical tension. Thus, apical constriction appears to be triggered not by a change in cortical tension, but by dynamic linking of apical cell-cell contact zones to an already contractile apical cortex.

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    01/03/12 | Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex.
    Ji N, Sato TR, Betzig E
    Proceedings of the National Academy of Sciences of the United States of America. 2012 Jan 3;109:22-7. doi: 10.1073/pnas.1109202108

    The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca(2+) imaging in single neurons.

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    11/18/11 | Facile and general synthesis of photoactivatable xanthene dyes.
    Wysocki LM, Grimm JB, Tkachuk AN, Brown TA, Betzig E, Lavis LD
    Angewandte Chemie. 2011 Nov 18;50:11206-9. doi: 10.1002/anie.201104571

    Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, “leuco” intermediates.

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