Fig. 4.

Acute inhibition of Plk1 during anaphase disrupts RhoA localization and blocks the onset of cytokinesis. (a and b) Metaphase Plk1^wt (a) and Plk1^as (b) cells were treated with 10 μM 3-MB-PP1 (time 0) and followed during progression into anaphase. Although chromatid separation occurred normally, Plk1^as cells failed to divide and became binucleated (SI Movies 1 and 2). (c) Plk1 activity is required to localize RhoA at the equatorial cortex. Plk1^wt and Plk1^as cells were synchronized with monastrol, released for 30 min, and then treated for 20 min with either blebbistatin or 3-MB-PP1. RhoA accumulation at the equatorial cortex was visualized after trichloroacetic acid fixation (21, 27). Anaphase cells (n = 100 per sample) were classified with respect to equatorial RhoA staining and cleavage furrow formation. (d–f) Plk1^wt and Plk1^as cells were treated with 3-MB-PP1 and stained with antibodies against phosphorylated myosin regulatory light chain (MRLC) (d), citron kinase (citron-K) (e), and anillin (f).