Figure S3.

MRCKβ is required for cup morphogenesis and particle internalization. (a–c) Confocal xy scanning analysis of Dbl3-depleted cells using two distinct MRCKβ siRNAs in primary porcine RPE cultures treated with POS-FITC for 1 h followed by 2-h chase. Cells were fixed and not permeabilized to detect external bound POS using anti–Annexin 5 antibody in addition to total POS-FITC. (d) siRNA-mediated knockdown of MRCKβ in primary porcine RPE cells leads to prolonged, longer, and disordered pseudopods with increased F-actin labeling. (e) Inhibition of MRCKβ kinase activity using BDP5290 results in a membrane remodeling defect in a similar manner to knock-down of MRCKβ. Note, POS is often misaligned and not centered at extended pseudopods in either MRCKβ siRNA-treated or BDP5290-treated cells, as indicated by green arrowheads in the confocal xy-scanning sections. (f) N-WASP and MRCK inhibitors in cells treated with POS for 30-min block clustering of mechanosensory talin. (g–i) Confocal xy analysis of integrin αvβ5 distribution in cups with or without MRCKβ inhibition or knockdown. Note, inhibition of MRCKβ-driven actomyosin contractility results in loss of integrin αvβ5 clustering along the deformed pseudopods that do not mature into normal cylindrical cups, as highlighted by F-actin staining. (j) pMLC and F-actin labeling increase during transition from nascent protrusion to cup formation. Scale bars represent 10 μm. All quantifications are based on n = 3 independent experiments and show the data points, means ±1SD, the total number of cells analyzed for each type of sample across all experiments, and P values derived from t tests. Source data are available for this figure: SourceData FS3.