Figure 4.

Phenotype of the for3DAD* mutant. (A) Immnunoblot of for3p-HA and for3pDAD*-HA expressed from the endogenous promoter. Twelve micrograms of total yeast extract were loaded in each lane. Levels were monitored with the TAT anti-α-tubulin antibody. (B) FRAP analysis of for3p-3GFP and for3pDAD*-2GFP. Using a laser scanning microscope, the entire cell tip of cells expressing either for3p-3GFP or for3pDAD*-2GFP from the endogenous promoter was photobleached and imaged every 4 s thereafter to monitor fluorescence recovery. Each trace represents the average value for the indicated number of experiments. Error bars represent the SE. (C) Single focal plane widefield fluorescence images of for3p-3GFP and for3pDAD*-2GFP expressed from the endogenous promoter. Note that for3pDAD* localizes to cell tips, if not even more tightly than wild type for3p. (D) Projection images of spinning disk confocal stacks of AlexaFluor 488-phalloidin stained wild-type (top) and for3DAD* (bottom) cells. Note that actin cables in the for3DAD* mutant stain more brightly than in wild-type cells. (E) Quantification of the fluorescence intensity of actin cables in wild-type and for3DAD* cells. We measured the peaks of the fluorescence profile along a line drawn across actin cables and subtracted background value. A histogram of these values is shown. Note that the fluorescence value is arbitrary and varies from one experiment to the other, but that under identical staining and imaging conditions, for3DAD* cells always show stronger actin cable staining than wild-type cells.