Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but not Itgb5-/- or Mfge8-/- mouse retina.

In the mammalian retina, life-long renewal of light-sensitive photoreceptor outer segments (POS) involves circadian shedding of distal rod POS tips and their subsequent phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Molecular mechanisms that promote or synchronize POS tip shedding have thus far remained unknown. Here we examined plasma membrane asymmetry of living POS by quantifying surface exposure of the membrane phospholipid phosphatidylserine (PS) using antibodies, annexin V, and pSIVA (polarity-sensitive indicator of viability and apoptosis), an annexin-based biosensor with switchable states of fluorescence. We found that isolated POS particles possess externalized PS, whose blockade or removal reduces their binding and engulfment by RPE in culture. Imaging of live photoreceptors in freshly dissected mouse retina detected PS externalization restricted to POS tips with discrete boundaries. In wild-type mice, frequency of rod tips exposing PS and length of tips with exposed PS peak shortly after light onset. In contrast, PS-marked POS tips do not vary in mice lacking the diurnal phagocytic rhythm of the RPE due to loss of either the phagocytosis receptor αvβ5 integrin, expressed by the RPE but not by photoreceptors, or its extracellular ligand milk fat globule-EGF factor 8 (MFG-E8). These data identify a molecular distinction, localized PS exposure, that is specific to the surface of rod POS tips. Enhanced PS exposure preceding rod shedding and phagocytosis suggests that surface PS promotes these processes. Moreover, our results demonstrate that the diurnal rhythm of PS demarcation of POS tips is not intrinsic to rod photoreceptors but requires activities of the RPE as well.