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      Control of plasma membrane lipid homeostasis by the extended synaptotagmins

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          Abstract

          Acute metabolic changes of plasma membrane (PM) lipids, such as those mediating signaling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the Extended-Synaptotagmins (E-Syts), ER proteins which function as PI(4,5)P 2 and Ca 2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro and such transfer requires Ca 2+ and their SMP domain, a lipid-harboring module. Genome edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but display enhanced and sustained accumulation of PM diacylglycerol (DAG) upon PI(4,5)P 2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syts-dependent ER-PM tethers in response to stimuli that cleave PI(4,5)P 2 and elevate Ca 2+ may help reverse accumulation of DAG in the PM by transferring it to the ER for metabolic recycling.

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          Most cited references 47

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          RNA-guided human genome engineering via Cas9.

          Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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            An ER-mitochondria tethering complex revealed by a synthetic biology screen.

            Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange.
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              A transcription activator-like effector toolbox for genome engineering.

              Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nat. Cell Biol.
                Nature cell biology
                1465-7392
                1476-4679
                16 March 2016
                11 April 2016
                May 2016
                11 October 2016
                : 18
                : 5
                : 504-515
                Affiliations
                [1 ]Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA
                [2 ]Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA
                [3 ]Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA
                [4 ]Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA
                [5 ]Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510, USA
                [6 ]Lipotype GmbH, Am Tatzberg 47-49, 01307 Dresden, Germany
                [7 ]Laboratoire de Physique Statistique, Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientifique, UMR 8550, 24 rue Lhomond, 75005 Paris, France
                [8 ]Present address: Lee Kong Chian School of Medicine, Nanyang Technological University, 308232, Singapore
                Author notes
                [*]

                equal contribution

                Article
                NIHMS768341
                10.1038/ncb3339
                4848133
                27065097

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                Cell biology

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