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Updated: Jun 26, 2026
Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Mertk drives myosin II redistribution during retinal pigment epithelial phagocytosis
David J Strick1, Wei Feng, Douglas Vollrath
1Department of Genetics, Stanford University, Stanford, California 94305-5120, USA.
Purpose:
Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis.
Methods:
Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells.
Results:
Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect.
Conclusions:
Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.
Insights
Mertk signaling mobilizes myosin II for photoreceptor outer segment ingestion in retinal pigment epithelial cells. This myosin II function is essential for clearing debris and preventing retinal degeneration.
Area of Science:
- Cell Biology
- Immunology
- Ophthalmology
Background:
- Mertk is a crucial phagocytic receptor in multiple systems, including vision.
- In the retinal pigment epithelium (RPE), Mertk mediates photoreceptor outer segment (OS) ingestion, vital for retinal health.
- Mertk dysfunction leads to retinal degeneration, but its precise role in RPE phagocytosis remains unclear.
Purpose of the Study:
- To identify proteins interacting with Mertk during OS phagocytosis.
- To elucidate the mechanism by which Mertk regulates RPE phagocytosis.
Main Methods:
- Immunoprecipitation of Mertk from RPE-J cells stimulated with OS.
- Mass spectrometry to identify interacting proteins.
- Confocal microscopy, pharmacologic inhibition, and siRNA knockdown in primary RPE cells to assess phagocytic function.
Main Results:
- Non-muscle myosin II-A (Myh9) was identified as a Mertk-interacting protein.
- Myosin II isoforms redistributed to ingested OS in wild-type RPE cells but not in Mertk-deficient cells.
- Inhibition or knockdown of myosin II impaired OS phagocytosis.
Conclusions:
- Mertk recruits myosin II from the RPE cell periphery to sites of OS engulfment.
- Myosin II is essential for the efficient phagocytic ingestion of OS by RPE cells.
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