Analysis of the linkage of MYRIP and MYO7A to melanosomes by RAB27A in retinal pigment epithelial cells

The apical region of the retinal pigment epithelium (RPE) typically contains melanosomes. Their apical distribution is dependent on RAB27A and the unconventional myosin, MYO7A. Evidence from studies using in vitro binding assays, melanocyte transfection, and immunolocalization have indicated that the exophilin, MYRIP, links RAB27A on melanosomes to MYO7A, analogous to the manner that melanophilin links RAB27A on melanocyte melanosomes to MYO5A. To test the functionality of this hypothesis in RPE cells, we have examined the relationship among MYRIP, RAB27A and MYO7A with studies of RPE cells in primary culture (including live-cell imaging), analyses of mutant mouse retinas, and RPE cell fractionation experiments. Our results indicate that the retinal distribution of MYRIP is limited to the RPE, mainly the apical region. In RPE cells, RAB27A, MYRIP, and MYO7A were all associated with melanosomes, undergoing both slow and rapid movements. Analyses of mutant mice provide genetic evidence that MYRIP is linked to melanosomes via RAB27A, but show that recruitment of MYRIP to apical RPE is independent of melanosomes and RAB27A. RAB27A and MYRIP also associated with motile small vesicles of unknown origin. The present results provide evidence from live RPE cells that the RAB27A-MYRIP-MYO7A complex functions in melanosome motility. They also demonstrate that RAB27A provides an essential link to the melanosome. Cell Motil. Cytoskeleton 64:474-487, 2007. (C) 2007 Wiley-Liss, Inc.