Pea2 recruits Myo2 to the cortex. (A) Upper panel: Length/width ratios of buds of WT (mean 1.07; n = 244), pea2Δ (0.88; n = 189), pea2_Δ286–330 (0.85; n = 260), myo2_RA (0.91; n = 234), and myo2_RD(0.89; n = 247) cells. Error bars indicate SD of the mean. Lower panel: DIC pictures of cells of the corresponding genotypes. Scale bar: 5 µm. (B) Representative fluorescence images of small budded WT or myo2_RD cells expressing GFP-Pea2, Myo2-GFP, Spa2-GFP, or Bud6-GFP. Scale bar: 5 µm. (C) Plot profile of the distribution of Myo2-GFP in WT (gray line, n = 12) or myo2_RDcells (blue line, n = 12). RFI of single cells was measured along the shortest distance from bud tip to neck (red arrow) and normalized to 1. Error bars indicate SD of the mean. (D) Quantification of GFP-Pea2 and Spa2-GFP fluorescence intensities along the cortex of WT (gray) and myo2_RD cells (blue). Each oval plot profile (red curved arrow in the upper cartoon) shows the mean of n = 12 single normalized profiles. Error bars indicate SD (light gray and blue) of the mean. (E) Disruption of the Pea2–Myo2 complex impairs Myo2’s association with the cortex. Left: FLIP analysis of Myo2-GFP in WT (n = 20), myo2_RD (n = 23), and pea2Δ cells (n = 32). Photobleaching of mother cells was done every 5 s, and z-stack images were taken every second. Curves are the fitted mean of single measurements. Right: Calculated FLIP half-times of Myo2-GFP in WT (33.8 s), myo2_RD (19.9 s), and pea2Δ cells (16.8 s). Error bars are SD. ns, not significant; ***, P < 0.001 by one-way ANOVA followed by a Kruskal-Wallis comparison test (A) or Turkey’s multiple comparisons test (E).