Polo-Like Kinase 1 Directs Assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF Complex to Initiate Cleavage Furrow Formation (original) (raw)
Figure 3
Plk1 phosphorylation of HsCyk-4 is essential for cleavage furrow formation.
(A) The indicated stable HeLa cell lines and control HeLa cells were transfected with siRNA to deplete endogenous HsCyk-4. At 32 h post-transfection, lysates were prepared, separated on SDS-PAGE, and probed with antibodies to HsCyk-4, GFP, and α-tubulin. (B) The indicated stable cell lines and control HeLa cells were transfected with siRNA to deplete endogenous HsCyk-4. 32 h post-transfection cells were fixed and stained with GFP and α-tubulin antibodies, and DNA was stained with DAPI. Failed cytokinesis events were scored from the percentage of bi- or multinucleated GFP-positive interphase cells relative to the total population of GFP-positive cells (_n_≥300). Error bars represent standard deviation from two independent experiments. (C) The indicated stable cells transfected with siRNA to deplete endogenous HsCyk-4 were filmed by live video microscopy and scored for the indicated phenotypes. Only those cells expressing HsCyk-4–EGFP with a maximum intensity value above 2,000 over background fluorescence were scored for phenotype. The total number of cells scored for each cell line: EGFP, n = 9; wt, n = 15; 4A, n = 32; 4D, n = 23. (D) Nomarski and GFP time-lapse images from (C) are shown from early anaphase (1 min post metaphase exit) through late telophase (13 min post metaphase exit) for the indicated stable cells lines. Because of high levels of expression, EGFP stable cells were filmed using reduced exposure times.