We explored the mechanisms and regulation of rhodopsin endocytosis in Drosophila by examining the trafficking itineraries of the major rhodopsin (Rh1) in vivo using transgenic flies expressing a modified Rh1 containing a C‐terminal mCherry tag (Rh1‐mCherry). By fluorescence microscopy, Rh1‐mCherry was detected in the visual organelles (rhabdomeres) of photoreceptors. Upon continued light stimulation, Rh1‐mCherry display light‐dependent endocytosis, and becomes enriched in the cytoplasm of photoreceptors. Internalization of rhodopsin may be mediated by the clathrin‐mediated endocytosis (CME). CME requires the presence of a minor phospholipid, phosphoinositol‐4, 5‐bisphosphate (PIP2) in the membrane. Interestingly, perturbation of the PIP2 biosynthesis fails to prevent the endocytosis of Rh1‐mCherry in rdgA mutants. The rdgA gene encodes diacylglycerol kinase critical for the recycling of PIP2.We also explored the contribution of two visual arrestins, Arr1 and Arr2, as adaptor proteins in promoting the endocytosis of Rh1. We introduced Rh1‐mCherry into either arr1 or arr2 mutant background and investigated how trafficking is affected. We show that internalization of Rh1‐mCherry is drastically reduced in the arr2 mutant. Arr2 is expressed 7fold more abundant than Arr1. Based on the finding, we conclude that Arr2 is critical for orchestrating the light‐dependent endocytosis of Rh1‐mCherry. Importantly, this Arr2‐mediated event does not appear to belong to the classical CME as it is insensitive to a reduction of PIP2 in the membrane.

Members of the conventional protein kinase C (cPKC) family are activated by both DAG and Ca2+ and have been implicated in the regulation of the actin cytoskeleton. Drosophila contains two cPKCs, Pkc53E (Pkc1) and eye-PKC (Pkc2); mutants missing each PKC lead to retinal degeneration. While eye-PKC is critical for the visual signaling, the role of Pkc53E is not known. We identified a photoreceptor-specific isoform of Pkc53E and show Pkc53E-RNAi negatively impacts the actin cytoskeleton of rhabdomeres. Interestingly, Pkc53E-RNAi enhances the degeneration of norpAP24 photoreceptors, suggesting Pkc53E could be activated independently of NorpA/PLCβ4. We further demonstrate that in norpAP24 photoreceptors Plc21C can be activated by Gq, which is responsible for the activation of Pkc53E. We explored whether Pkc53E regulates adducin in Drosophila photoreceptors. Adducin cross-links the actin cytoskeleton to the spectrin network, which is blunted by PKC phosphorylation. Importantly, we observed that phosphorylation of adducin was greatly reduced in a null allele of pkc53E. Downregulation of hts that encodes Drosophila adducin, exerts a more severe effect than Pkc53E-RNAi to impact the actin cytoskeleton. In contrast, overexpression of a mCherry tagged Add2, one of the three Drosophila adducin isoforms, led to the apical expansion of rhabdomeres with overgrowth of the actin cytoskeleton. This phenotype is likely caused by the dominant-negative activity of the tagged Add2 as it also was observed in α-spectrin-RNAi or β-spectrin-RNAi. Interestingly, downregulation of Pkc53E does not suppress the expansion of rhabdomeres during development, but negatively affects the appearance of rhabdomeres in adult photoreceptors. We conclude that Drosophila adducin has two distinct functions: in pupal photoreceptors, it regulates rhabdomere morphogenesis, which is independent of Pkc53E. In adult photoreceptors, it promotes the maintenance of the actin cytoskeleton, which is regulated by Pkc53E in response to the light-induced activation of the PLCβ activity.

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