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Fig. 1. Bud6p-GFP localisation is perturbed in formin mutants. (A) Representative images of wild-type cells or the indicated mutants expressing Bud6p-GFP at endogenous level and CFP-Tub1p. MT structures provided an independent landmark for cell cycle progression. Cells prior to spindle assembly (a), cells with <1 µm (b) or <2 µm (c) long spindles, and cells with elongated spindles (d) are shown. Relative to wild-type cells, a bnr1 mutation delayed labelling of the bud neck, whereas both bni1 or bni1^CT mutations advanced Bud6p accumulation at the bud neck relative to spindle assembly. A bnr1 also delayed Bud6p-GFP accumulation in combination with bni1^CT. Images are 2D projections of five-plane z-stacks. Scale bar: 2 µm. (B) Bud6p-GFP accumulation as a ring at the bud neck relative to spindle length. At least 161 budded cells (excluding cells at mitotic exit) were scored in digital images. Error bars indicate s.e.m. (C,D) Accumulation of Bud6p-GFP at the bud neck is decreased in a bnr1 mutant relative to wild-type cells. (C) Maximal fluorescence intensity at the bud tip or bud neck as a function of bud length in wild-type versus bnr1 cells. Data were collected by six-pixel-wide linescan analysis in digital images of 64 wild-type or bnr1 cells as depicted in the diagram. Broken lines indicate arbitrary boundaries for small-, medium- and large-budded cells. (D) Mean fluorescence intensity was measured within a fixed 10x5 pixels region at the bud neck or within the entire bud, excluding the bud neck (see diagram) in 20 wild-type or bnr1 mid-size budded cells (bud length between 1.5 and 3.0 µm). Error bars indicate s.e.m.

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