Fig. 7. Mlc1, Spa2 and FM4-64 colocalise to a surface crescent structure in pseudohyphal buds. (A-F) Pseudohyphae were induced in strains carrying the indicated fusions to YFP or stained with FM4-64. Cells were stained with Calcofluor white (A-C) and with filipin (D). The images shown are a projection of the deconvolved Z-stack, except C, which is a single layer. Numbers in panels A and B refer to cells discussed in the text. (D) A pseudohyphal cell expressing Mlc1-YFP and stained with filipin. Left, deconvolved image; right, model generated with the Softworx^TM suite. (E and F) Localisation of Spa2-YFP and FM4-64, respectively, in pseudohyphae. Arrows in F indicate crescents of FM4-64 staining at the tips of pseudohyphal buds. (G) Mlc1-YFP-expressing pseudohyphal cells were sampled at various times after inoculation and fixed and stained with DAPI. Cells were categorised into bins of bud length and characterised according to whether Mlc1-YFP formed a ball or a crescent at the tip or was not present at all. In addition, cells that contained two nuclei were categorised as having completed mitosis. Note that the categories at each bud length do not add up to 100% as cells can be in more than one category. For example, a cell can be categorised as having no Mlc1 at the tip as well as having a cytokinetic ring. Each bin contains at least 100 cells.