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. 2007 Oct;18(10):4155–4167. doi: 10.1091/mbc.E07-02-0094

Figure 1.

Figure 1.

Cdc42p is necessary for cell morphology, actin cable organization and for3p localization. (A) Growth curve of wild-type and cdc42-1625 cells incubated at 28 and 36°C. (B) Rescue of the temperature-sensitive growth defect of cdc42-1625 by different cdc42 alleles. cdc42-1625 cells were transformed with the expression plasmid pREP41-HA carrying either no insert, wild-type cdc42+, constitutively active cdc42G12V, or dominant-negative cdc42T17N alleles and grown on Edinburgh minimal media plates for 2 d at 25 and 36°C. (C) Immunoblot of HA-cdc42p and HA-cdc42p-1625 expressed from the endogenous promoter. Twenty-five micrograms of total yeast extracts from exponential cultures at 25 and 36°C were loaded in each lane. Levels were monitored with the anti-actin antibody. Although wild-type cdc42p shows increased levels at 36°C, this increase failed to happen in the cdc42-1625 mutant. (D) Immunofluorescence of HA-cdc42p and HA-cdc42p-1625 with anti-HA antibody. Both wild-type and mutant cdc42p localize to growing cell tips. Differential interference contrast (DIC) of the permeabilized cells is shown at the bottom. (E) Morphology of wild-type and cdc42-1625 cells grown to log phase at 28 and 36°C for 5 h. DIC and calcofluor-staining images are shown. (F) Rescue of cell morphology (DIC; top) and actin cables (AlexaFluor-phalloidin; bottom) by wild-type cdc42+ in cdc42-1625 cells at 36°C. (G) Projection images of spinning disk confocal stacks of AlexaFluor 488-phalloidin–stained cdc42-1625 (top) and wild-type (bottom) cells grown at 25°C (left) or 36°C for 1 h (right). Note that actin cables are extremely weak, but they were still present in cdc42-1625 cells (arrowheads). (H) Single focal plane widefield fluorescence images of myo52p-tomato in cdc42-1625 (top) and wild-type (bottom) cells grown at 25°C. (I) Single focal plane widefield fluorescence images of for3p-3GFP in cdc42-1625 (top) and wild-type (bottom) cells grown at 25°C. Note that for3p largely fails to localize to cell tips in the mutant cells. All bars, 2 μm.