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      Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

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          Abstract

          Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments 1 or require light and are difficult to use 2 . Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed 3, 4 . MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.

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          Computer visualization of three-dimensional image data using IMOD.

          We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
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            Site-directed mutagenesis by overlap extension using the polymerase chain reaction

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              Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.

              Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca(2+) uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca(2+) uniporter.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                16 May 2013
                21 October 2012
                November 2012
                02 July 2013
                : 30
                : 11
                : 1143-1148
                Affiliations
                [1 ]Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [2 ]National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, University of California at San Diego, La Jolla, California, USA
                [3 ]Departments of Systems Biology and Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, USA
                [4 ]Broad Institute, Cambridge, Massachusetts, USA
                [5 ]Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California, USA
                [6 ]Department of Neurosciences, University of California at San Diego, La Jolla, California, USA
                Author notes
                Correspondence should be addressed to A.Y.T. ( ating@ 123456mit.edu )
                Article
                NIHMS463908
                10.1038/nbt.2375
                3699407
                23086203
                a62bd91c-8a1c-4b5d-b5ff-38015b47fd8e

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                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM072881 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM065937 || GM
                Funded by: National Center for Research Resources : NCRR
                Award ID: P41 RR004050 || RR
                Funded by: National Center for Research Resources : NCRR
                Award ID: P41 RR002250 || RR
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: P41 GM103412 || GM
                Categories
                Article

                Biotechnology
                Biotechnology

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