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First published online 28 October 2008
doi: 10.1242/jcs.036269

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Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

View larger version (39K): [in this window] [in a new window]   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 bni1CT mutations advanced Bud6p accumulation at the bud neck relative to spindle assembly. A bnr1 also delayed Bud6p-GFP accumulation in combination with bni1CT. 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.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Actin organisation in formin mutants. Analysis of F-actin structures in fixed cells of asynchronous populations stained by rhodamine-phalloidin. (A) Representative images for actin organisation in the indicated strains. Bar, 2 µm. (B) Distribution of budded cells from asynchronous populations (excluding cells at cytokinesis), according to the presence of polarised actin cables and patches, as depicted in the diagrams: thick or numerous cables directed towards the neck and polarised patches (1), thin or few cables directed towards the neck and polarised patches (2), disorganised cables but polarised patches (3), no visible cable but polarised patches (4), no visible cable and depolarised patches (5). Over 400 cells were scored. Error bars indicate s.e.m. (C,D) Bni1CTp significantly supports cell polarity. Diploid budding pattern scored as an indirect indicator of actin organisation and cell polarity in wild-type cells or the indicated mutants. (C) Representative images of fixed diploid cells stained with calcofluor to label chitin-containing scars illustrating bipolar (left) or random (right) budding pattern. Bar, 2 µm. (D) Distribution of diploid cells according to their budding pattern (n=500 cells). bni1/BNI1 and bni1/bni1CT exhibited comparable bipolar budding relative to a bni1/bni1. Error bars indicate s.e.m. (E,F) Analysis of actin organisation for synergism effects. (E) Synergistic loss of actin organisation and cell polarity in bud6 bni1 cells. (F) Synergism is not apparent between bud6 and bni1CT bnr1 mutations. Cells were scored as indicated in B.

View larger version (37K): [in this window] [in a new window]   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 bni1CT 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, bni1CT 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 bni1CT 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 Bni1CTp-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.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Bud61-565p restored microtubule capture but not actin organisation to a bud6 strain. (A) Representative images for localisation of GFP-Bud6p or GFP-Bud61-565p expressed under the HIS3 promoter in bud6 cells. GFP-Bud61-565p was efficiently localised to the bud and bud neck. Images are 2D projections of five-plane z-stacks. Bar, 2 µm. (B,C) Actin organisation defects of bud6 were not rescued by expression of Bud61-565p. (B) Representative images of fixed cells stained with rhodamine-phalloidin showing actin organisation in bud6 cells expressing Bud6p or Bud61-565p. Bar, 2 µm. (C) Quantitation of actin organisation for the indicated strains as in Fig. 2; n>400 cells. Error bars indicate s.e.m. (D) MT–cell cortex interactions scored in time lapse series of the indicated strains during the interval from mitotic exit to bud emergence as in Fig. 4C. At least 140 cortical interactions were scored per strain. Expression of full-length Bud6p or Bud61-565p restored MT shrinkage in a bud6 mutant (P<0.0001) to the level observed in wild-type cells. (E) Correct preanaphase spindle orientation along the mother bud axis was scored (Theesfeld et al., 1999; Huisman et al., 2004) in the indicated strains showing that bud61-565 suppressed the genetic interaction between kar9 and bud6. (F) Spindle polarity in anaphase cells (only one SPB in contact with the bud) (Huisman et al., 2004) was scored in the indicated strains, showing that bud61-565 suppressed the genetic interaction between dyn1 and bud6.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Bud6p and Bud61-565p sediment with taxol-stabilised microtubules. (A,B) In vitro MT-binding assays were carried out using whole cell extracts from bud6 cells expressing either HA3-Bud6p (A) or HA3-Bud61-565p (B). Extracts were incubated with taxol-stabilised microtubules (Taxol-MTs), free tubulin in the presence of nocodazole (Noco-Tub) or buffer (–) as a control. Following centrifugation through a 10% (A) or 20% (B) sucrose cushion, supernatant and pellet (MT) fractions were assayed by western blot analysis. (C) MT fractions obtained as in A were subsequently resuspended in one of the following: 8 mM ATP, 10 mM GTP, 0.5 M NaCl or 2 M urea and subjected to sedimentation through a second sucrose cushion. Pellet and supernatant fractions were analysed by western blotting. Only urea treatment decreased Bud6p association to MTs. (D) Bud61-565p sedimented in association with MTs from whole cell extracts of bni1CT bnr1 cells.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Contributions of formins to spindle orientation revealed by genetic analysis. The impact of mutating formin genes on actin cable organisation (red lines) Bud6p accumulation (green circles), actin-mediated transport (black arrowheads) and spindle position (green bar) is depicted schematically. For each mutant, an integrative overview is provided, including (A) sites for Bud6p-dependent MT capture, (B) preferred target sites for Kar9p-mediated delivery, and the differential contributions of Bud6p and Kar9p as deduced from the respective mutant phenotypes (C,D) in otherwise wild-type cells as well as in combination with formin mutations (Miller et al., 1999; Lee et al., 1999; Segal et al., 2000a, Yeh et al., 2000; Huisman et al., 2004). For simplicity, the additive effects of mutating BUD6 or KAR9 are not depicted in the diagrams.

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