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568104.fig.007b
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Figure 7: Macropinocytosis and clathrin localize in PDGF-BB-induced circular ruffles. (a) Visualization using ESEM of macropinocytic structures in cells that were stimulated with PDGF-BB. ESEM studies revealed the formation of macropinocytic cups ((a)-A and (a)-B) and membrane blebs on the dorsal membrane ((a)-A and (a)-D). Membrane blebs co-localized with circular ruffles (a)-A but were also localized on other parts of the dorsal membrane (a)-D. The membrane blebs that did not co-localize with circular ruffles were larger and might result from the closure of dorsal circular ruffles (a)-D. The smaller membrane blebs that did co-localize with circular ruffles (a)-A may represent macropinosomes that result from the fission of small ruffles that are formed on top of circular ruffles as represented in (a)-C. Imaging conditions of A, B, C: HV = 6 kV at 100%RH. Image D: HV = 3 kV (to reveal more surface detail) at 100%RH. A, B: bar represents 50 μm, C, D: bar represents 10 μm. (b) Cells exhibit macropinocytic vesicles in dorsal circular ruffles. Circular ruffles were identified in PDGF-BB stimulated cells by staining for F-actin (b)-B. Circular ruffles exhibit macropinocytic vesicles (arrows). The macropinosome is enriched with phosphorylated PDGF β-receptors (b)-A. Bar represents 10 μm. (c) Clathrin localizes in newly formed circular ruffles upon PDGF-BB stimulation in mouse fibroblasts. Serum-starved cells exhibit abundant stress fibers (c)-B, and clathrin is randomly distributed in the cytoplasm of serum-starved cells (c)-A. Upon stimulation with PDGF-BB, circular ruffles are induced by actin dynamics (c)-E and clathrin co-localizes with F-actin in the newly formed circular ruffles (c)-D. Bar represents 10 μm.