Dataset Information

The Proteome of Native Adult Muller Glial Cells From Murine Retina.

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 H2O2-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.

SUBMITTER: Grosche A

PROVIDER: S-EPMC4739667 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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

Grosche Antje A Hauser Alexandra A Lepper Marlen Franziska MF Mayo Rebecca R von Toerne Christine C Merl-Pham Juliane J Hauck Stefanie M SM

Molecular & cellular proteomics : MCP 20150831 2

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 pro ...[more]

PMID: 26324419

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Project description:The multiplex, lattice mosaic of cone photoreceptors in the adult fish retina is a compelling example of a highly ordered epithelial cell pattern, with single cell width rows and columns of cones and precisely defined neighbor relationships among different cone types. Cellular mechanisms patterning this multiplex mosaic are not understood. Physical models can provide new insights into fundamental mechanisms of biological patterning. In earlier work, we developed a mathematical model of photoreceptor cell packing in the zebrafish retina, which predicted that anisotropic mechanical tension in the retinal epithelium orients planar polarized adhesive interfaces to align the columns as cone photoreceptors are generated at the retinal margin during post-embryonic growth.With cell-specific fluorescent reporters and in vivo imaging of the growing retinal margin in transparent juvenile zebrafish we provide the first view of how cell packing, spatial arrangement, and cell identity are coordinated to build the lattice mosaic. With targeted laser ablation we probed the tissue mechanics of the retinal epithelium.Within the lattice mosaic, planar polarized Crumbs adhesion proteins pack cones into a single cell width column; between columns, N-cadherin-mediated adherens junctions stabilize Müller glial apical processes. The concentration of activated pMyosin II at these punctate adherens junctions suggests that these glial bands are under tension, forming a physical barrier between cone columns and contributing to mechanical stress anisotropies in the epithelial sheet. Unexpectedly, we discovered that the appearance of such parallel bands of Müller glial apical processes precedes the packing of cones into single cell width columns, hinting at a possible role for glia in the initial organization of the lattice mosaic. Targeted laser ablation of Müller glia directly demonstrates that these glial processes support anisotropic mechanical tension in the planar dimension of the retinal epithelium.These findings uncovered a novel structural feature of Müller glia associated with alignment of photoreceptors into a lattice mosaic in the zebrafish retina. This is the first demonstration, to our knowledge, of planar, anisotropic mechanical forces mediated by glial cells.

Dataset Information

The Proteome of Native Adult Muller Glial Cells From Murine Retina.

Publications

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

Similar Datasets

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