Phagocytic Cup Assembly During Rod Outer Segment Phagocytosis By The Retinal Pigment Epithelium
Diurnal phagocytosis of spent photoreceptor outer segment tips (POS) by retinal pigment epithelial (RPE) cells is essential for life-long retinal function. Clearance of POS is triggered by engagement of &agr;vβ5 integrin and Mer tyrosine kinase (MerTK) surface receptors of the RPE by their corresponding extracellular ligands. POS engulfment requires F-actin phagocytic cup formation. These studies presented in this dissertation sought to identify Rho GTPases family proteins that are involved in the regulation of this F-actin phagocytic cup formation during POS phagocytosis by RPE cells. I found that Rac1 is activated while RhoA-ROCK is inhibited during RPE phagocytosis. The activation of Rac1 is a necessary response to POS stimulation preceding POS engulfment. Rac1 is not activated by POS in RPE lacking &agr;vβ5 but occurs normally in RPE lacking MerTK in vivo and in culture. In contrast, RhoA-ROCK activity is down-regulated during phagocytosis by wt RPE but not by MerTK-deficient RPE. Lack of MerTK or its ligand also results in failure of phagocytic cup formation and engulfment. I then tested the dependence of F-actin recruitment to bound particles on Rac1 and the RhoA-ROCK pathway when MerTK signaling pathway is silent. To this end, I investigated the F-actin distribution and phagocytic activity of RPE cells in culture fed with POS with or without ligand supplementation and activating or inhibiting Rac1 and RhoA/ROCK either genetically or pharmacologically. Surface-bound POS failed to recruit F-actin and were not engulfed in the absence of MerTK signaling unless either Rac1 was constitutively and in excess active or RhoA/ROCK was inhibited. ROCK inhibition did not restore F-actin recruitment or engulfment abolished by expression of dominant-negative Rac1 and, conversely, constitutively-active Rac1 failed to restore F-actin recruitment or engulfment inhibited by activation of RhoA. Neither pathway manipulation affected the activity of the other pathway. Taken together, these results demonstrate that activation of Rac1 and inhibition of RhoA-ROCK must both contribute to productive F-actin dynamics that is required for POS engulfment by RPE cells.
Molecular biology|Cellular biology|Ophthalmology
Mao, Yingyu, "Phagocytic Cup Assembly During Rod Outer Segment Phagocytosis By The Retinal Pigment Epithelium" (2015). ETD Collection for Fordham University. AAI3728400.