PI 3-kinase independent role for AKT in F-actin regulation during outer segment phagocytosis by RPE cells

Abstract

Daily phagocytosis of photoreceptor outer segment fragments (POS) by the retinal pigment epithelium (RPE) is essential for vision. RPE cells use an uptake machinery that is highly similar to the one macrophages use to phagocytose apoptotic cells. In both forms of phagocytosis, particle binding induces phagocyte signaling that is required for F-actin assembly and re-arrangement beneath bound particles. Macrophage binding of apoptotic cells stimulates PI3 kinases (PI3K) and AKT kinases (AKT), which may be downstream of PI3K, and PI3K inhibition decreases engulfment. Here, we used specific inhibitory agents to investigate whether and how PI3K and AKT contribute to RPE phagocytosis. Either PI3K or AKT inhibition eliminated AKT activation by RPE cells in response to POS and increased the numbers of POS bound by RPE cells. Analyzing the quality of bound POS, we found a higher fraction of POS associated with F-actin phagocytic cups and myosin II in RPE receiving AKT inhibitor. In these cells, individual POS also recruited more F-actin and myosin II than POS in control cells. In contrast, PI3K inhibition did not alter frequency of phagocytic cups but individual cups contained less F-actin (but similar levels of myosin II) compared to control cups. Annexin AII, another phagocytic cup protein of RPE cells, associated with bound POS regardless of inhibitor treatment. POS engulfment proceeded normally if cells already carried surface-bound POS when receiving inhibitors. However, PI3K inhibition during POS binding blocked subsequent POS engulfment. In striking contrast, AKT inhibition had no effect on POS engulfment. Taken together, these results suggest distinct regulatory roles of PI3K and AKT during POS phagocytosis by RPE cells.