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A. Hitt, L.W. Schovan, D.C. Dziedzic, S.D. Laing, J.R. Reddan; A Lipid Raft Fraction from Cultured Lens Epithelial Cells Contains a Putative Ponticulin Homologue . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1250.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Signal transduction pathways are often initiated at the plasma membrane by signaling molecules residing in lipid rafts, lipid microdomains. We find that lipid rafts are present in cultured lens epithelial cells. Since signal transduction pathways can be mediated by components of the actin cytoskeleton, we identified F-actin binding proteins in cell lysates and in the detergent insoluble lipid raft fraction. Methods: The lens epithelial cell lines N/N1003A and FHL124 were routinely cultured, scraped and the triton-insoluble floating fraction (TIFF) was isolated. A newly developed Alexa F-actin blot overlay assay (Hitt, et al, 2002, Anal. Biochem. 310:67-71) was used to identify functional F-actin binding proteins present in membranes and lipid raft domains of human and rabbit lens epithelial cells. Results: Interactions between the plasma membrane and the cytoskeleton are thought to be responsible for the control of many fundamental processes including signal transduction. The major high-affinity link between the cortical actin network and the plasma membrane in the model system D. discoideum has been identified as ponticulin, a 17-kDa transmembrane component of lipid rafts. Ponticulin is one of a very few integral membrane proteins that interact directly with the F-actin-based cytoskeleton and the only transmembrane protein known to nucleate F-actin in vitro. Experiments were designed to determine if a ponticulin homologue exists in lens epithelial cells. F-actin binding activity was detected in two molecular weight ranges, approximately 75-kDa and 17-kDa in membrane fractions of both cell lines. In addition, a 17-kDa -actin binding activity was detected in the TIFF or lipid raft fraction of the FHL124 cells. The F-actin binding activity of the 17-kDa band is likely to be a functional homologue of D. discoideum ponticulin and may play a role in signal transduction. Conclusions: Data show that this 17-kDa F-actin binding protein is present in both rabbit (N/N1003A) and human (FLH124) lens epithelial cells. In addition there is a 17-kDa F-actin binding protein present in lipid rafts of human (FHL124) lens epithelial cells. Current studies are directed towards the identification and subcellular localization of these F-actin binding proteins. A proteomic analysis of lipid raft proteins may permit identification of changes in signaling pathways associated with cataract formation and aging.
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