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D. J. Strick, W. Feng, D. Vollrath; Non-Muscle Myosin II Functions in RPE Phagocytosis Through Interaction With the Mertk Receptor. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3979.
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Phagocytosis of rod outer segments (OS) by the retinal pigment epithelium (RPE) is essential for the viability of mammalian photoreceptors. Loss of RPE phagocytic function due to mutation of the Mertk gene leads to accumulation of OS debris and ultimately death of rod and cone photoreceptors in RCS rats and Mertk knockout mice. Furthermore, MERTK mutations in humans have also been shown to cause retinitis pigmentosa. Previous studies have shown that the Mertk receptor is directly involved in ingestion of rod OS in vitro and that gene transfer of wild-type Mertk rescues the phagocytic defect of RCS RPE both in vitro and in vivo. However, little is known about how Mertk specifically regulates OS ingestion. In order to address this question, we searched for proteins that bound directly or indirectly to Mertk during OS phagocytosis.
Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Immunoprecipitated Mertk complexes were fractionated by SDS-PAGE and identified by mass spectrometry. Candidates were characterized by using confocal microscopy and either pharmacologic inhibition or siRNA knockdown coupled with an in vitro phagocytosis assay in rat primary RPE cells.
Mass spectrometry analysis of a 220 kDa band enriched in OS treated samples identified Myh9, the non-muscle myosin II-A heavy chain. Using immunofluorescence microscopy, we demonstrated that both myosin II-A and II-B isoforms redistribute in rat primary RPE cells and colocalize with ingested OS over time. Rat primary RPE cells treated with the myosin II specific inhibitor blebbistatin showed a significant phagocytic defect when compared to control cells. Furthermore, siRNA targeted against the non-muscle myosin II-A or II-B heavy chain genes also inhibited RPE phagocytosis of OS. Preliminary studies indicate that phosphorylation of the myosin regulatory light chain at Ser19 is diminished in RPE/choroid tissue harvested from Mertk knockout mice.
Our results demonstrate that myosin II-A and II-B function cooperatively during RPE phagocytosis of OS, and suggest that Mertk interacts with and activates non-muscle myosin II, either directly or indirectly, to enable extension and/or closure of phagocytic cups.
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