Fig. 5.

Mechanostress reverses alpha-factor induced cell polarization by inhibiting Fus3 activity in a Pkc1-dependent manner. (A) Redistribution of the yeast actin cytoskeleton of cells polarized with alpha-factor for 90 min and exposed to mechanostress for 30 min. The actin cytoskeleton was stained with rhodamine phalloidin. The number of actin patches in the cell body exposed (+) or not (−) to mechanostress was quantified after deconvolution, and shown as box−whisker plots, which depict 5th, 25th, 50th, 75th, and 95th percentiles of actin foci. At least 50 cells were analyzed for each experiment. (B) Redistribution of Bni1 away from shmoo tips after mechanostress. Wild-type or mid2Δ cells expressing Bni1-qV were exposed to alpha-factor for 90 min, and the polarized cells were subjected to mechanostress. Maximum intensity projected images of cells prior to (−) and after 15 min of mechanostress (+) are shown. (C) Quantification of images from B. The dynamics of Bni1-qV under mechanostress was quantified in terms of dispersal from the shmoo tip and recruitment to the plasma membrane away from the shmoo tip. The percentage of cells that dispersed or recruited Bni1 is depicted in the graph. The experiment was performed three times with at least 50 cells counted for each condition. (D) Dynamics of Bni1-qV with mechanostress at the indicated conditions was quantified as in C. (E) Analog-sensitive fus3-as1 cells expressing Bni1-qV were polarized with alpha-factor for 90 min, and Bni1-qV redistribution was monitored after inhibiting Fus3 activity with 1Na-PP1 for 20 min. (F) Quantification of Bni1 dispersal away from shmoo tips upon mechanostress. The fraction of cells that dispersed more than 25% of Bni1, quantified by measuring the Bni1 intensity at the 4 pixel × 4 pixel brightest area is represented for each condition. The experiment was performed three times, and at least 50 cells were counted for each condition. A Student’s t test was performed and the statistical significance is indicated [not significant (n.s.) = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001]. Images are taken with objective lens (magnification: B and E, 60×).