Figure 2. Inhibition of dynamic actin assembly or disassembly blocks endocytic uptake via the GEEC pathway.

A) Control (right panel) or Lat A-treated (left panel) FRαTb cells were incubated for 2 min at 37°C with either A568-Tf and PLF (top panel) or A568-Tf and FITC-dextran (lower panel) and imaged at high resolution. Grayscale images of PLF/FITC-dextran (green) and A568-Tf-labelled TfR (red)-containing structures were pseudocoloured and colour merged. Scale bar, 10 μm. B) Histogram shows the extent of uptake after a 5-min pulse of the indicated endocytic tracers in cells subject to Lat A, Jas or Cyto D treatment as described in Materials and Methods, expressed relative to that measured in control cells (value of controls set to 1). Values represented are wt. mean ± SEM obtained across three experiments. C) Histogram shows the average number of GEECs estimated in cells treated with different actin poisons as compared with control cells (value of controls set to 1). Values represented are wt. mean ± SEM across two experiments. D) Histogram shows that regurgitation of fluid is inhibited upon depletion of cholesterol or treatment of cells with Lat A. FRαTb cells were treated with MβCD or Lat A or carrier and pulsed with F-Dex for 2 min and chased for indicated times. Cells were fixed and amount of probe associated with the cells was estimated. Fluid left after the chase was normalized to the amount internalized (initial uptake set to 1). Values represented are wt. mean ± SEM across two experiments.