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      ATG2 transports lipids to promote autophagosome biogenesis

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          Abstract

          Valverde et al. show that the autophagy protein ATG2 functions in autophagosome biogenesis by transferring lipids at ER–autophagosome contact sites.

          Abstract

          During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER–autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.

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          Most cited references 36

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          Genome engineering using the CRISPR-Cas9 system.

          Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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            MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

            MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.
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              Automated electron microscope tomography using robust prediction of specimen movements.

               D Mastronarde (2005)
              A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                jcb
                The Journal of Cell Biology
                Rockefeller University Press
                0021-9525
                1540-8140
                28 June 2019
                05 April 2019
                : 218
                : 6
                : 1787-1798
                Affiliations
                [1 ]Department of Cell Biology, Yale University School of Medicine, New Haven, CT
                [2 ]Laboratory of Molecular Electron Microscopy, The Rockefeller University, New York, NY
                Author notes
                Correspondence to Thomas J. Melia: thomas.melia@ 123456yale.edu
                [*]

                D.P. Valverde and S. Yu contributed equally to this paper and the first author was selected by a coin flip.

                Article
                201811139
                10.1083/jcb.201811139
                6548141
                30952800
                © 2019 Valverde et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

                Page count
                Pages: 12
                Product
                Funding
                Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002,%22National%20Institutes%20of%20Health%22;
                Award ID: GM1000930
                Award ID: NS063973
                Award ID: GM080616
                Award ID: GM114068
                Funded by: China Scholarship Council, DOI https://doi.org/10.13039/501100004543;
                Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002;
                Award ID: T32 GM007223
                Funded by: National Science Foundation, DOI https://doi.org/10.13039/100000001;
                Categories
                Research Articles
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                Cell biology

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