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196 Publications

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    01/06/20 | Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells
    Schumann B, Malaker SA, Wisnovsky SP, Debets MF, Agbay AJ, Fernandez D, Wagner LJ, Lin L, Li Z, Choi J, Fox DM, Peh J, Gray MA, Pedram K, Kohler JJ, Mrksich M, Bertozzi CR
    Molecular Cell. Jan-06-2020;78(5):824 - 834.e15. doi: 10.1016/j.molcel.2020.03.030

    Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

     
     

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    01/06/20 | Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.
    Zhang Y, Schroeder LK, Lessard MD, Kidd P, Chung J, Song Y, Benedetti L, Li Y, Ries J, Grimm JB, Lavis LD, De Camilli P, Rothman JE, Baddeley D, Bewersdorf J
    Nature Methods. 2020 Jan 06;17(2):225-231. doi: 10.1038/s41592-019-0676-4

    Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

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    01/06/20 | Probabilistic Models of Larval Zebrafish Behavior Reveal Structure on Many Scales
    Robert Evan Johnson , Scott Linderman , Thomas Panier , Caroline Lei Wee , Erin Song , Kristian Joseph Herrera , Andrew Miller , Florian Engert
    Current Biology. 01/2020;30:70 - 82.e4. doi: https://doi.org/10.1016/j.cub.2019.11.026

    Nervous systems have evolved to combine environmental information with internal state to select and generate adaptive behavioral sequences. To better understand these computations and their implementation in neural circuits, natural behavior must be carefully measured and quantified. Here, we collect high spatial resolution video of single zebrafish larvae swimming in a naturalistic environment and develop models of their action selection across exploration and hunting. Zebrafish larvae swim in punctuated bouts separated by longer periods of rest called interbout intervals. We take advantage of this structure by categorizing bouts into discrete types and representing their behavior as labeled sequences of bout types emitted over time. We then construct probabilistic models—specifically, marked renewal processes—to evaluate how bout types and interbout intervals are selected by the fish as a function of its internal hunger state, behavioral history, and the locations and properties of nearby prey. Finally, we evaluate the models by their predictive likelihood and their ability to generate realistic trajectories of virtual fish swimming through simulated environments. Our simulations capture multiple timescales of structure in larval zebrafish behavior and expose many ways in which hunger state influences their action selection to promote food seeking during hunger and safety during satiety.

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    01/03/20 | The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila.
    Agrawal P, Kao D, Chung P, Looger LL
    Journal of Experimental Biology. 2020 Jan 03;223(2):. doi: 10.1242/jeb.207407

    Social isolation strongly modulates behavior across the animal kingdom. We utilized the fruit fly to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide (), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of neurons each similarly increased isolation-driven aggression. Our results suggest a U-shaped dependence of social isolation-induced aggressive behavior on signaling, similar to the actions of many neuromodulators in other contexts.

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    01/02/20 | Tissue clearing and its applications in neuroscience.
    Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ
    Nature Reviews Neuroscience. 2020 Jan 02;21(2):61-79. doi: 10.1038/s41583-019-0250-1

    State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

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    01/01/20 | Gas cluster ion beam SEM for imaging of large tissue samples with 10 nm isotropic resolution.
    Hayworth KJ, Peale D, Januszewski M, Knott GW, Lu Z, Xu CS, Hess HF
    Nature Methods. 2020 Jan 01;17(1):68-71. doi: 10.1038/s41592-019-0641-2

    We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with <10 nm isotropic resolution of each section, and these can then be stitched together to span the sectioned volume. Incorporating gas cluster ion beam SEM into existing single-beam and multibeam SEM workflows should be straightforward, increasing reliability while improving z resolution by a factor of three or more.

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