Figure 4.

Dyn1 I533A targets to but fails to mediate fission of CCPs. (A) Top panels, live cell epifluorescence imaging of retrovirus-infected Dyn2 KO mouse fibroblasts expressing Tomato-clathrin light chain along with GFP fusion constructs of either Dyn1 WT or Dyn1 I533A. Scale bar, 10 μm. Bottom panels, magnified images from insets on top panels that demonstrate colocalization of clathrin and dynamin. (B) Top, EM classification of CCP profiles according to degree of membrane curvature and invagination. Classes I and II represent shallow, whereas classes III and IV represent deeply invaginated coated pits that remain tethered to the plasma membrane via a narrow membrane neck. Scale bar, 100 nm. Bottom, percent occurrence of these classes in tTA-HeLa cells overexpressing either Dyn1 WT (□) or Dyn1 I533A (■) or neither (▧) is plotted. Greater than 100 CCP profiles were scored for each sample.