Figure 5.

FIP3 and Rab11 regulate the late stage of cytokinesis. (A) HeLa cells were transfected with FIP3 siRNA and incubated for 48 h. Cells were then harvested and levels of FIP3 protein determined by immunoblotting. (B) HeLa cells were transfected with FIP3 siRNA and incubated for 48 or 72 h before they were fixed and stained with anti-FIP3 antibodies and DAPI stain. The number of cells with one, two, or more than two nuclei were then counted and expressed as a percentage of the total number of cells. The numbers on top of the figure indicate the total number of cells counted for each condition. (C–E) HeLa cells were either mock-transfected (C) or transfected with FIP3 (D) or Rab11 (E) siRNAs. Cells were incubated for 72 h, fixed, and stained with anti-FIP3 (C and D, green), anti-Rab11 (E, green), or antitubulin (C–E, red) antibodies. Nuclei were visualized using DAPI stain (D and E, blue). (F) Mock-transfected HeLa cells (also see Supplementary Movie 1) were imaged using phase-contrast time-lapse microscopy. Cells in the metaphase were chosen for analysis and images were collected every 1.4 min for 2.5 h. (G) HeLa cells transfected with FIP3 siRNA were incubated for 72 h and then imaged using time-lapse microscopy (also see Supplementary Movie 2). Cells in metaphase were chosen for analysis and images were collected every 1.4 min for 2.5 h. Arrowheads mark plasma membrane protrusions during telophase. An asterisk marks a cytoplasmic bridge between daughter cells. (H) HeLa cells transfected with FIP3 siRNA were incubated for 72 h and then imaged using time-lapse microscopy (also see Supplementary Movie 3). Cells attempting cytokinesis were imaged using phase-contrast time-lapse microscopy. Images were collected every 1.4 min for 4.5 h. During most of time-lapse series cells remained connected by a narrow cytoplasmic bridge (asterisk) and did not undergo abscission. At the end of the time-lapse series, cells broke the cytoplasmic bridge (arrow) and separated.