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Fig. 5. A fraction of Sec15p localizes in an actin-independent fashion. Yeast cells were grown in SC-Ura overnight to early log phase. For GFP visualization, cells were harvested, fixed in methanol, permeabilized in acetone, rehydrated in PBS, and imaged in both fluorescence and differential interference contrast (DIC) modes. Actin polarization was monitored by staining cells with FITC-conjugated phalloidin. (A) An overnight culture of act1-3 cells carrying Sec15-GFP as the sole copy of Sec15p (Sec15GFP; NY2556) was shifted to 37°C for 90 minutes (or incubated at 20°C) prior to methanol fixation. The localization was examined by direct fluorescence microscopy and the proportion of cells containing a polarized GFP signal was scored (right). At least 200 cells were scored in each strain. (B) Wild-type yeast cells expressing GFP-tagged Sec15 (Sec15GFP; NY2557) were grown to early log phase and treated either with DMSO alone or with 200 µM latrunculin A (LatA) in DMSO. Following treatment for 15 minutes, cells were rapidly fixed by methanol/acetone and prepared for microscopy. The localization was examined by direct fluorescence microscopy and the proportion of cells containing a polarized GFP signal was scored (right). At least 200 cells were scored in each strain. (C) The localization of Sec15-GFP (Sec15GFP) in wild-type cells (NY2557) exiting G0 at 25°C in the absence or presence of latrunculin A was examined at time points of 1, 2 and 3.5 hours. (D) The localization of Sec8-GFP (Sec8GFP; NY2558) in wild-type cells exiting G0 was examined and the cells were observed after 3.5 hours of incubation in the absence or presence of latrunculin A (Lat-A). (E) Quantification of the GFP fluorescence to assess the percentage of cells incubated in the absence or presence of Lat-A that exhibited polarized Sec15-GFP (Sec15GFP) or Sec8-GFP (Sec8GFP) proteins. At least 200 cells were scored in each strain.
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