Arp2/3 Inhibition Suppresses Membrane Dynamics but Does Not Block Pigment Granule Migration in Isolated Fish Retinal Pigment Epithelial (RPE) Cells.

Abstract

The retinal pigment epithelium (RPE) lies at the back of the vertebrate eye and plays important roles in vision. In the eyes of fish, which lack dilatable pupils, RPE pigment granules undergo massive migrations to regulate light intensity. Previous work has demonstrated that both aggregation and dispersion of pigment granules through apical projections of RPE require an intact actin cytoskeleton. To determine the role of the actin-nucleating complex, Arp2/3, on pigment granule movement, isolated RPE cells were treated with the inhibitors CK-666 and CK-869. Both drugs caused a reversible rapid cessation of both lateral and distal membrane extension of the cells' apical projections, resulting in an overall thinning of the projections. CK-666 also stimulated the formation of F-actin aggregates in projections, mainly at the distal tips. Despite these structural changes, these inhibitors had no effect on pigment granule aggregation or dispersion. Immunolocalization of Arp3 showed diffuse fluorescence throughout isolated cells. The actin side-binding protein, tropomyosin, was also present in RPE cells, aligning with F-actin bundles. While it cannot be ruled out that these drugs only partially inhibited Arp2/3 complex nucleation, the results suggest that while actin nucleation by Arp2/3 inhibition is important in membrane dynamics of the RPE apical projections, it is not critical for pigment granule movement.