Figure 3.

Membrane Targeting of ECT2’s GEF Domain Does Not Support Cytokinesis

(A) Domain organization of transgenic C1B proteins.

(B) Live-cell imaging of ECT2-C1B and GEF-C1B proteins in metaphase (upper panel) and GEF-C1B in anaphase (lower panel). Cells stably expressing the indicated transgenes were transfected with H2B-mCherry (red) and imaged 48 hr after transfection. Cells were treated with TPA at t = 0 min. The scale bars in this and the following panels represent 10 μm.

(C) IF analysis of cells stably expressing GEF-C1B. DMSO or TPA was added to the cells 6 hr after transfection with ECT2 siRNA. Multi-nucleation was analyzed 48 hr after transfection (n > 300 cells each from three independent experiments).

(D) IF analysis of anillin in metaphase cells expressing the indicated transgenes. Cells were treated with nocodazole to enrich the population for prometaphase cells. One hour after nocodazole washout, the cells were treated with DMSO or TPA for 5 min and analyzed. Membrane localization of ECT2 variants is largely lost upon cell fixation.

See also Figure S3.