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Fig. 7. Polarized F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, poles and septum, but not for its stable maintenance at the growing zones. (A) Maintenance of Bgs4p to the medial ring and poles does not require the F-actin function. Early log-phase cells (RFP-bgs4⁺ bgs4 crn1⁺-GFP) grown on YES liquid medium at 28°C were transferred to YES medium containing Lat A (100 µM) or an equal volume of DMSO as control and incubated at 28°C for 15 minutes. Cells grown in the presence of DMSO displayed the same RFP-Bgs4p and Crn1p-GFP localization as initial control cells (data not shown). The Lat A-treated cells were washed, transferred to fresh YES medium, and incubated at 28°C for 3 hours. Cells were collected at the indicated times, fixed, and analyzed for RFP (Bgs4p) and GFP (actin-associated Crn1p) fluorescence. (B) The F-actin cytoskeleton is needed for Bgs4p relocalization to the growing regions. Cells grown as in A were exposed to stress conditions (high temperature and the absence of nitrogen) for 2 hours to induce Bgs4p and F-actin patch delocalization. Then, the cells were transferred to YES medium and incubated at 28°C for 2 hours in the presence of Lat A or DMSO (–Lat A control). Cells were collected at the indicated times and analyzed as in A. (C) The F-actin cytoskeleton is necessary for Bgs4p delocalization from the growing zones upon stress treatment. Cells were grown and treated with Lat A as in A to induce actin depolymerization and to maintain Bgs4p polarization. Then, the cells were washed and exposed to stress conditions (absence of nitrogen) in the presence of Lat A or DMSO (–Lat A control). Cells were collected at the indicated times and analyzed as in A.

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