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236 Publications

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    Gonen Lab
    09/01/14 | High-resolution structure determination by continuous-rotation data collection in MicroED.
    Nannenga BL, Shi D, Leslie AG, Gonen T
    Nature Methods. 2014 Sep;11(9):927-30. doi: 10.1038/nmeth.3043

    MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

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    08/22/14 | Light sheet-based imaging and analysis of early embryogenesis in the fruit fly.
    Khairy K, Lemon WC, Amat F, Keller PJ
    Methods in Molecular Biology. 2015;1189:79-97. doi: 10.1007/978-1-4939-1164-6_6

    The fruit fly is an excellent model system for investigating the sequence of epithelial tissue invaginations constituting the process of gastrulation. By combining recent advancements in light sheet fluorescence microscopy (LSFM) and image processing, the three-dimensional fly embryo morphology and relevant gene expression patterns can be accurately recorded throughout the entire process of embryogenesis. LSFM provides exceptionally high imaging speed, high signal-to-noise ratio, low level of photoinduced damage, and good optical penetration depth. This powerful combination of capabilities makes LSFM particularly suitable for live imaging of the fly embryo.The resulting high-information-content image data are subsequently processed to obtain the outlines of cells and cell nuclei, as well as the geometry of the whole embryo tissue by image segmentation. Furthermore, morphodynamics information is extracted by computationally tracking objects in the image. Towards that goal we describe the successful implementation of a fast fitting strategy of Gaussian mixture models.The data obtained by image processing is well-suited for hypothesis testing of the detailed biomechanics of the gastrulating embryo. Typically this involves constructing computational mechanics models that consist of an objective function providing an estimate of strain energy for a given morphological configuration of the tissue, and a numerical minimization mechanism of this energy, achieved by varying morphological parameters.In this chapter, we provide an overview of in vivo imaging of fruit fly embryos using LSFM, computational tools suitable for processing the resulting images, and examples of computational biomechanical simulations of fly embryo gastrulation.

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    Ji LabGENIE
    08/17/14 | Multiplexed aberration measurement for deep tissue imaging in vivo.
    Wang C, Liu R, Milkie DE, Sun W, Tan Z, Kerlin A, Chen T, Kim DS, Ji N
    Nature Methods. 2014 Aug 17:. doi: 10.1038/nmeth.3068

    We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

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    08/15/14 | ChIP for hox proteins from Drosophila imaginal discs.
    Agrawal P, Shashidhara LS
    Methods in Molecular Biology. 2014 August 15;1196:241-53. doi: 10.1007/978-1-4939-1242-1_15

    Chromatin immunoprecipitation (ChIP) is a technique that reveals in vivo location of a protein bound to DNA. ChIP coupled with DNA microarrays (ChIP-chip) or next-generation sequencing (ChIP-seq) allows for identification of binding sites of transcription factors on a global scale. Here we describe a protocol for ChIP to identify binding of the Ultrabithorax (Ubx) Hox transcription factors from imaginal discs of Drosophila larvae. The protocol can be extended to other model organisms and transcription factors.

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    08/15/14 | Large environments reveal the statistical structure governing hippocampal representations.
    Rich PD, Liaw H, Lee AK
    Science. 2014 Aug 15;345(6198):814-7. doi: 10.1126/science.1255635

    The rules governing the formation of spatial maps in the hippocampus have not been determined. We investigated the large-scale structure of place field activity by recording hippocampal neurons in rats exploring a previously unencountered 48-meter-long track. Single-cell and population activities were well described by a two-parameter stochastic model. Individual neurons had their own characteristic propensity for forming fields randomly along the track, with some cells expressing many fields and many exhibiting few or none. Because of the particular distribution of propensities across cells, the number of neurons with fields scaled logarithmically with track length over a wide, ethological range. These features constrain hippocampal memory mechanisms, may allow efficient encoding of environments and experiences of vastly different extents and durations, and could reflect general principles of population coding.

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    08/14/14 | Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy.
    Guo M, Ehrlicher AJ, Jensen MH, Renz M, Moore JR, Goldman RD, Lippincott-Schwartz J, Mackintosh FC, Weitz DA
    Cell. 2014 Aug 14;158(4):822-32. doi: 10.1016/j.cell.2014.06.051

    Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

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    Gonen Lab
    08/13/14 | A type VI secretion-related pathway in bacteroidetes mediates interbacterial antagonism.
    Russell AB, Wexler AG, Harding BN, Whitney JC, Bohn AJ, Goo YA, Tran BQ, Barry NA, Zheng H, Peterson SB, Chou S, Gonen T, Goodlett DR, Goodman AL, Mougous JD
    Cell Host Microbe. 2014 Aug 13;16(2):227-36. doi: 10.1016/j.chom.2014.07.007

    Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

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    08/07/14 | A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.
    Li H, Kroll JR, Lennox SM, Ogundeyi O, Jeter J, Depasquale G, Truman JW
    Cell Reports. 2014 Aug 7;8(3):897-908. doi: 10.1016/j.celrep.2014.06.065

    We report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.

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    08/05/14 | Cytoplasmic tyrosine phosphatase Shp2 coordinates hepatic regulation of bile acid and FGF15/19 signaling to repress bile acid synthesis.
    Li S, Hsu DD, Li B, Luo X, Alderson N, Qiao L, Ma L, Zhu HH, He Z, Suino-Powell K, Ji K, Li J, Shao J, Xu HE, Li T, Feng G
    Cell Metab. 08/2014;20(2):320-32. doi: 10.1016/j.cmet.2014.05.020

    Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR) and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that nonreceptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through the orchestration of multiple signals in hepatocytes.

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