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      Directed evolution of APEX2 for electron microscopy and proteomics

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          Abstract

          APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.

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          Most cited references23

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          MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake

          Mitochondrial calcium uptake plays a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients, and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here, we utilize an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics, and organelle proteomics. RNA interference against 13 top candidates highlighted one gene that we now call mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the organelle’s inner membrane and has two canonical EF hands that are essential for its activity, suggesting a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high capacity mitochondrial calcium entry. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.
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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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              Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

              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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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                31 October 2014
                24 November 2014
                January 2015
                01 July 2015
                : 12
                : 1
                : 51-54
                Affiliations
                [1 ]Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139
                [2 ]Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114
                [3 ]National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, CA, 92093
                [4 ]Department of Neurosciences, University of California at San Diego, La Jolla, CA, 92093
                Author notes
                Correspondence should be addressed to A.Y.T. ( ating@ 123456mit.edu )
                Article
                NIHMS639105
                10.1038/nmeth.3179
                4296904
                25419960
                228fca6e-fc8d-4799-861f-cf15f85faa25
                History
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                Life sciences
                Life sciences

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