The order of appearance of Myo2 and Pea2 at bud neck and presumptive bud site. (A) Time-lapse analysis of cells coexpressing GFP-Pea2 and Myo2-mCherry in WT cells. Arrows indicate the first visible GFP or mCherry signal occurring during cytokinesis at the bud neck (minutes 3 and 8) and in G1 during assembly of the presumptive bud site (minute 20). Scale bar: 3 µm. (B) Appearance of Myo2-mCherry (Myo2_tx) compared with GFP-Pea2 at the presumptive bud site and bud neck in WT and myo2_RD cells. Myo2 and Pea2 appear simultaneously at the presumptive bud site in WT cells (0.02 min, n = 53) and myo2_RD cells (−0.11 min, n = 54). During cytokinesis, Myo2-mCherry appears 4.87 min after GFP-Pea2 at the bud neck of WT cells (n = 52) and 5.23 min after GFP-Pea2 at the bud neck of myo2_RD cells (n = 53). Error bars are SD. ns, not significant (one-way ANOVA followed by Tukey’s multiple comparison test).