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Fig. 6. ROCK and actomyosin inhibitors disrupt the polarized distribution of clathrin at the uropod. (A) The effects of Rho/ROCK and myosin II inhibitors on the distribution of membrane CSs (green circles) and on the cell morphology during integrin-mediated cell adhesion. (B) Density of CSs/µm² – labeled with GFP-ClLC or endogenous -adaptin – at the basal plasma membrane (adhesion plane) of cells pre-incubated with Y-27632 (1 hour), blebbistatin (30 minutes) or cytochalasin D (1 hours). More than 1000 CSs from 10 representative cells were visualized for each inhibitor and labeling, except the control cells, in highly magnified images. Frequency of CSs at the basal membrane of control cells was minimal. The distribution of AP-2 is shown in supplementary material Fig. S2E. (C) Distribution of GFP-ClLC (green) in a representative control cell. The adhesion plane is pointed at the lateral view (arrow). Basal membrane was simultaneously stained with the membrane marker FM 4-64 (red); note how the frequency of CSs is very low in control cells compared with the plasma membrane red stain. (D) Distribution of CSs in cells plated on fibronectin and pre-treated with C3 (14 hours), Y-27632 (1 hour), or blebbistatin (30 minutes). Panels show the endogenous clathrin heavy chain (ClHC) (C3) and GFP-ClLC (Y-27632, blebbistatin) labeling at the basal membrane. Enlarged areas are indicated by blue boxes, and DIC images merged with nuclear DAPI (blue) are shown. Bars, 5 µm.

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