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Fig. 3. Analysis of formin involvement in Bud6p-dependent cortical capture. (A) Distribution of astral MT-cortex interactions by cell compartment (see diagram) scored in time lapse series during the preanaphase interval. At least 350 interactions by 200 MTs per strain were scored. Error bars indicate s.e.m. (B) Selected frames from time-lapse series of wild-type, bnr1 or bni1^CT cells expressing GFP-Tub1p, illustrating characteristic astral MT distribution prior to anaphase onset. Compared with wild-type cells, astral MTs in bnr1 cells reached more often into the bud. Conversely, bni1^CT cells favoured astral MT contacts with the bud neck. Time elapsed in seconds relative to onset of spindle elongation (t=0). Scale bar: 2 µm. (C) Distribution of MT–cortex interactions from mitotic exit to bud emergence in the daughter cell. Categories are: (a) shrink, MTs shortening while in continued contact with the cell cortex; and (b) growth, MTs growing while in continued contact with the cell cortex; (c) hit, MT transient contacts with the cortex during one cycle of MT polymerization and depolymerisation without any associated SPB movement; and (d) sweep and sliding, MT + end movement while in contact with the cell cortex. (a) and (b) are accompanied by SPB movement towards or away from a fixed point of contact, respectively. Over 200 cortical interactions were scored in 10 cells. Error bars indicate s.e.m. Wild-type and bnr1 bni1^CT cells exhibited comparable frequencies of MT shrinkage at the cell cortex. Deletion of BUD6 significantly decreased this frequency (P<0.0001). (D) Fluorescence images corresponding to 2D-projections of five-plane z-stacks showing Bni1^CTp-GFP correct localisation in otherwise wild-type, bud6 or bud6 bnr1 backgrounds, respectively. Scale bar: 2 µm. (E) Preanaphase spindle position (2 µm-long spindles) in the indicated strains (n=500). Categories are: spindle close to the bud neck but misaligned (blue), spindle close to the bud neck and aligned (red), spindle within the bud (yellow) and spindle within the distal (away from the bud neck) half of the mother cell (green). Error bar and s.e.m.

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