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The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how [...]

Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting.

Purpose: To map the expression and distribution of FGFR and potential FGFR-related signaling molecules within rat and human retina., Methods: Sections of postnatal 5 day old and adult rat, and aged human retina, and cell cultures prepared from selected cell populations of young rat retina, were immunolabeled with specific antisera to FGFR (FGFR-1, -2, -3, and -4) or candidate signaling molecules [phospholipase Cg1 (PLCg1), son of sevenless 1 and 2 (SOS1, SOS2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein tyrosine phosphatase (SH-PTP2) and SH2-containing protein (Shc)], and with multiple retinal cell-type specific antibodies. Controls were conducted using primary antisera pre-adsorbed with the corresponding immunizing peptide., Results: All FGFR antisera showed strong labeling of inner retina [inner nuclear layer, inner plexiform layer and ganglion cell layer (INL, IPL and GCL respectively)] in rat and human retina, although there were distinct differences in individual patterns. FGFR-3 was particularly intense in ganglion cell bodies and dendrites, and was absent from photoreceptors and bipolar cells in vitro. FGFR-1 and FGFR-4 also labeled the outer nuclear layer (ONL), more intensely in adult than in young tissue, and FGFR-4 was especially prominent within inner segments. FGFR-2 and -3 were only weakly expressed in the ONL, but FGFR-2 showed specific labeling of cone outer segments in human retina. Candidate FGFR-signaling molecules also showed generally higher expression in the inner than outer retina in the different samples. Shc immunolabeling was apparent in the GCL and nascent photoreceptor outer segments in young and adult retina. SOS1 expression was much more intense than SOS2 in the ONL, although the latter showed selective intense staining of a sub-population in the INL and GCL. These ex vivo data were confirmed in cultures prepared from young rat retina. Pure photoreceptor cultures exhibited strong expression of FGFR-1 and -4, and faint expression of FGFR-2 and -3. In mixed inner retinal cultures, anti-FGFR-1 labeled neurons and Müller glia with equal intensity, while the other FGFR antisera showed preferential staining of neurons. FGFR-3 was strongly expressed by ganglion and amacrine cells but not by other types. Signaling molecules showed widespread expression, but of variable intensity, in all cells. All control experiments using corresponding peptide pre-adsorption led to complete removal of immunostaining., Conclusions: Rat and human retinal cells showed a largely similar, widespread expression of multiple FGFR and candidate FGFR-related signaling molecules. Distinct differences in development, species, cell- and sub-cell type distribution were apparent, suggesting that specific FGFR/FGF ligands and transduction pathways may operate in different cells.