Figure 6

Mutations in Ser354 or Arg344 abolish the dominant negative effect of POB1-PRD1 on EGF endocytosis. a) HeLa cells were transiently transfected with GFP-POB1 PRD1, GFP-POB1 or GFP-Dynamin K44, serum starved and incubated with TRITC-EGF for 20 minutes at 37°C. Cells were fixed and observed on an Olympus IX 70 with Nanomover^® and softWoRx DeltaVision. GFP transfected cells are visible in the 528 nm channel (green). TRITC EGF was visualized at 617 nm (fluorescent spots). Nuclei were visualized by DAPI staining at 457 nm (blue). Transfected cells that do not show endocytic vescicles are indicated by a white arrow. b) HeLa cells were transiently transfected with GFP-POB1 PRD1 S354A or GFP-POB1 PRD1 R344A and treated as above. Transfected cells are visible in the 528 nm channel as green cells. These cells show a wild type distribution of fluorescent endocytic vesicles (TRITC-EGF visualized at 617 nm). Nuclei were visualized by DAPI staining at 457 nm (blue). c) Overexpression of 14-3-3 abolish the dominant negative effect of POB1-PRD1 on EGF endocytosis. HeLa cells were transiently transfected with GFP-POB1 PRD1 together with 3xFLAG-14-3-3 ζ. Cells were treated as above. Cells were then permeabilized and treated with Cy5-conjugated antibodies targeting the FLAG epitope of the 14-3-3 proteins, visible at 685 nm. Cells were observed as described above. These cells show a wild type distribution of fluorescent endocytic vesicles (TRITC-EGF visualized at 617 nm).