Role of Caveolin-1 in Phagolysosomal Digestion by The Retinal Pigment Epithelium
Retinal pigment epithelial (RPE) cells form a polarized monolayer directly beneath the light-sensitive outer segments of photoreceptors in the eye. RPE cells are responsible for the daily clearance of photoreceptor outer segment fragments (POS) shed in a circadian, diurnal rhythm by photoreceptors. This daily phagocytic function is essential for vision. Phagocytosis processes include three distinct yet coupled phases; recognition/ binding, engulfment, and phagolysosomal digestion. In this dissertation, I investigated if the membrane-organizer protein caveolin-1 plays a role in RPE clearance phagocytosis. Quantifying phagocytosis in the eye, I found that RPE cells in mice lacking caveolin-1 only in the RPE showed a normal phagocytic burst after light onset but retained excess phagosomes 8 hours later. Quantifying phagocytosis by RPE cells in culture I found that over-expression of wild-type caveolin-1 or a mutant caveolin-1 that functions normally anywhere but not at the plasma membrane did not affect recognition or engulfment but significantly accelerated POS digestion. Furthermore, caveolin-1 localized to acidified POS-opsin phagolysosomes. Digestion of POS-opsin, which constitutes ~90% of POS-protein, depends on the protease cathepsin D and deglycosylase β-N-Acetylglucosaminidase. Cathepsin D protein levels and activity and β-N-Acetylglucosaminidase activity in RPE lacking caveolin-1 were significantly lower following light onset than 8 hours later, while the opposite was observed for wild-type RPE. However, cathepsin D localization to phagosomes was independent of caveolin-1. In cells with excess and reduced levels of caveolin-1, the lysosomal pH was lower and higher, respectively. Finally, the activity of the lysosomal H + translocator, vATPase, was decreased in cells expressing reduced levels of caveolin-1. Taken together, these results suggest a novel function for caveolin-1 in RPE clearance phagocytosis that alters lysosomal pH subsequently altering lysosomal enzyme activity. Cholesterol-dependant membrane microdomains facilitate signaling processes by assembling signaling complexes. RPE phagocytosis employs integrin &agr;vβ5 and its co-receptor CD81 for POS binding, and FAK and MerTK activities for POS engulfment. Here, I found that perturbing membrane cholesterol leads to reduction in POS binding and ingestion, which may be affected indirectly. Perturbing membrane cholesterol also altered &agr;vβ5 integrin and CD81 membrane anchorage. Finally, cell culture and tissue analyses showed that CD81 anchorage depends on β5 integrin. Taken together, these results show that cholesterol-dependent microdomains are important for RPE phagocytosis and that CD81 requires integrin β5 for membrane microdomain anchorage.
Molecular biology|Cellular biology
Sethna, Saumil Sudhir, "Role of Caveolin-1 in Phagolysosomal Digestion by The Retinal Pigment Epithelium" (2014). ETD Collection for Fordham University. AAI3684488.