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      The Proteome of Native Adult Müller Glial Cells From Murine Retina*

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          Abstract

          To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes ( e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H 2O 2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies — a topic still controversially discussed.

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          Adhesion signaling - crosstalk between integrins, Src and Rho.

          Interactions between cells and the extracellular matrix coordinate signaling pathways that control various aspects of cellular behavior. Integrins sense the physical properties of the extracellular matrix and organize the cytoskeleton accordingly. In turn, this modulates signaling pathways that are triggered by various other transmembrane receptors and augments the cellular response to growth factors. Over the past years, it has become clear that there is extensive crosstalk between integrins, Src-family kinases and Rho-family GTPases at the heart of such adhesion signaling. In this Commentary, we discuss recent advances in our understanding of the dynamic regulation of the molecular connections between these three protein families. We also discuss how this signaling network can regulate a range of cellular processes that are important for normal tissue function and disease, including cell adhesion, spreading, migration and mechanotransduction.
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            Neuroscience. Developmental refining of neuroglial signaling?

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              A Mass Spectrometric-Derived Cell Surface Protein Atlas

              Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
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                Author and article information

                Journal
                Mol Cell Proteomics
                Mol. Cell Proteomics
                mcprot
                mcprot
                MCP
                Molecular & Cellular Proteomics : MCP
                The American Society for Biochemistry and Molecular Biology
                1535-9476
                1535-9484
                February 2016
                31 August 2015
                31 August 2015
                : 15
                : 2
                : 462-480
                Affiliations
                [1]From the ‡Insitute of Human Genetics, University of Regensburg, D-93053 Regensburg, Germany;
                [2]§Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany
                Author notes
                ¶ To whom correspondence should be addressed: Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany. Tel.: +49-89-3187-3941; Fax: +49-89-3187-4426; E-mail: hauck@ 123456helmholtz-muenchen.de ; Institute of Human Genetics, University of Regensburg, D-93053 Regensburg, Germany. Tel.: +49-941-944-5429; Fax: +49-941-944-5402; E-mail: antje.grosche@ 123456klinik.uni-regensburg.de .
                Author information
                http://orcid.org/0000-0003-0338-7530
                http://orcid.org/0000-0002-4132-4322
                http://orcid.org/0000-0002-3422-4083
                http://orcid.org/0000-0002-1630-6827
                Article
                M115.052183
                10.1074/mcp.M115.052183
                4739667
                26324419
                1c5f7849-2903-4167-aa9f-45e9e8020f3d
                © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                History
                : 1 June 2015
                : 14 August 2015
                Funding
                Funded by: Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659
                Award ID: HA 6014/2–2
                Categories
                Special Issue Articles

                Molecular biology
                Molecular biology

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