The role of annexin 2 in RPE phagocytosis of photoreceptor outer segments

The Retinal Pigmented Epithelium (RPE) has many functions, one of which is the phagocytosis of shed photoreceptor outer segments (POS). This process is vital to the maintenance of both the RPE and photoreceptors. Outer segment shedding and internalisation are under circadian regulation, such that shedding is followed by a burst of phagocytosis at the onset of light. Annexin 2 is well placed to have a role in this process. Its direct involvement in actin dynamics and association to vesicle membranes during endocytosis may be significant in RPE outer segment phagocytosis, as this process requires extensive re-organisation of actin and re-distribution of membrane on the apical processes of the RPE. This thesis examines cell differentiation in two RPE cell lines and in primary porcine RPE cells, in order to evaluate the best system for conducting phagocytosis experiments. In vitro experiments provided evidence that annexin 2 localises to the phagocytic cup during POS internalisation but dissociates once internalisation is complete. Following knock down of annexin 2, phagocytosis was shown to decrease. Furthermore annexin 2 was shown to be phosphorylated during phagocytosis. We also found that c-Src is phosphorylated alongside annexin 2 and therefore may phosphorylate annexin 2, which contains a c-Src phosphorylation site. To investigate the circadian aspects of POS phagocytosis by the RPE, apical and basal phagosomes were quantified in the RPE from annexin 2 knock out and wild type eyes, harvested before and after light onset. Phagosomes from eyes harvested one hour after light onset were also mapped relative to Bruch’s membrane. The annexin 2 knock out animals lack the characteristic burst of phagocytosis one hour after light onset exhibited in wild type animals. Phagosomes were also retarded in the apical processes one hour after light onset, at the peak of phagocytosis, when they are normally internalised into the cell body for processing and degradation. Lysates from wild type eyes showed that annexin 2 is phosphorylated before light onset along with c-Src and FAK, key molecules in the RPE phagocytic machinery. Importantly, the absence of annexin 2 in knock out eyes delayed phosphorylation of c-Src and FAK. This delay in phosphorylation of two key RPE phagocytosis molecules may account for the delay in ingestion of outer segments into the cell body and the accumulation of phagosomes in the apical processes observed in the knock out animals. In conclusion, work in this thesis has demonstrated that annexin 2 is required for efficient RPE internalisation of rod outer segments both in vitro and in vivo. Annexin 2 is required for the timely phosphorylation of FAK and c-Src, which may account for the delay in POS internalisation observed in the annexin 2 knock out mice