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A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

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      Abstract

      Mitochondrial Ca 2+ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival 1 , but the molecular identity of the Ca 2+ channel, the mitochondrial calcium uniporter 2 , was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator 3 , coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca 2+ uptake. MCU overexpression doubled the [Ca 2+] mt rise evoked by IP 3-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively-charged residues of the putative pore forming region were replaced, had no channel activity and reduced agonist-dependent [Ca 2+] mt transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca 2+ uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance.

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      Most cited references 32

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        A guided tour into subcellular colocalization analysis in light microscopy.

        It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.
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          Calcium signalling: dynamics, homeostasis and remodelling.

          Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.
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            Author and article information

            Affiliations
            [1 ]Department of Biomedical Sciences, University of Padua, Italy
            [2 ]Department of Biology, University of Padua, Italy
            [3 ]CNR Institute of Neuroscience, University of Padua, Italy
            Author notes
            Correspondence to: Rosario Rizzuto, Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy, Phone: +390498276061, Fax: +390498276049, rosario.rizzuto@ 123456unipd.it
            [*]

            These authors contributed equally to this work

            Journal
            0410462
            6011
            Nature
            Nature
            Nature
            0028-0836
            1476-4687
            23 January 2014
            19 June 2011
            18 August 2011
            22 August 2014
            : 476
            : 7360
            : 336-340
            21685888 4141877 10.1038/nature10230 NIHMS304276
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