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Fig. 10. Quantification of Mlc1 fluorescence in hyphae and pseudohyphae. Strains expressing either Mlc1-YFP or Spa2-YFP were cultured under the indicated conditions and the average peak fluorescence intensity was determined. An example of the 3D surface contour map of fluorescence is shown alongside the hyphal (A,D) or pseudohyphal (B) tip from which it was derived. (A) A ridge of lesser fluorescence surrounds a focused point of high fluorescence. (B) The contour map reveals only a structure whose maximum fluorescence resembles that of the ridge detected in A and no focal point of fluorescence. The histograms in A and B show the mean maximum fluorescence intensity in cells divided into bins according to bud length. Each bin consists of at least 100 cells. The time course experiments described in panels A and B have been carried out on three separate occasions with similar results. (C) Average peak fluorescence was compared between Spa2-YFP (black) and Mlc1-YFP (grey) in the different morphologic forms. Spa2 shows little variation in its peak intensity between growth forms whereas Mlc1 fluorescence is more than four times greater in hyphae (H) than pseudohyphae (PH) or yeast (Y). (D) The shape of the Spa2 fluorescence surface contour map in hyphae resembles that of Mlc1 in pseudohyphae shown in Panel B. Bar, 1 µm.
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