Fig 2: DLC2 and Kif1B regulate spindle positioning and microtubule growth.(a) Hela cells stably expressing mCherry-lifeact and GFP-α-tubulin were transfected with siRNAs and plated onto micropatterned dishes with parallel fibronectin lines. Time-lapse recordings were then made collecting images every minute to follow actin behaviour and spindle orientation (see Supplementary Movies 4–6 and Supplementary Fig. 7). The movies were then quantified by calculating the percentages of cells with a specific actin behaviour based on kymographs and spindle orientation (Pol, polarized actin; Circ, circular movement of actin; Abs, absent actin polarization). Blue bars represent cells with mitotic spindles aligned with the fibronectin line; red bars, misaligned spindles. Significance was calculated between corresponding subgroups (shown are means±1 s.d.; n=3 experiments). (b–e) HeLa cells stably transfected with GFP-EB3 and transfected with the indicated siRNAs were filmed every 2 s. Microtubule dynamics was then quantified in at least 10 cells, following 10 plus-ends per cell (see Supplementary Movies 7–9). The box-and-whisker plots show total distance tracked (b) and total time tracked (c) (n=45 microtubule tips). Microtubule tips sliding along the cell cortex were also quantified in 10 cells per type of siRNA transfection (d). The still images in panel e, taken every 2 s, show examples of microtubule tips tracked. Scale bars, 1 (upper panel in e) and 2 μm (lower panels in e). (*P<0.05, ***P<0.001; analysis of variance).

Fig 3: DLC2 and Kif1B regulate mDia3 recruitment at the cell cortex.(a,b) Control, DLC2- and Kif1B-depleted cells were stained for mDia3. b shows a quantification of mDia3 staining at the lateral cell cortex (means±1 s.d.; n=10 cells). Note: cortical mDia3 staining is elevated when Cdc42 activity is increased and cortical Kif1B levels are low (see Fig. 4). (c) Quantification of cells with bipolar spindles with aligned and misaligned metaphase plates transfected with the indicated siRNAs (means±1 s.d.; n=3; at least 60 mitotic cells were counted per condition in each of these three experiments). Scale bars, 10 μm. (**P<0.01, ***P<0.001; t-test).

Fig 4: DLC2 and Kif1B regulate the formation of bipolar spindles after monastrol release.Confocal microscopy pictures for control, DLC2 and Kif1B, Cdc42, mDia3 and Cdc42/DLC2 siRNA-transfected HCE cells treated with monastrol. Cells were stained for HURP (a) and PCR-1 (b). The charts show the length of astral (HURP) and kinetochore (PCR-1) microtubules in HCE cells treated with monastrol (shown are means±1 s.d.; n=3 experiments). Scale bars, 10 μm. (*P<0.05, **P<0.01, ***P<0.001; t-test.)

Fig 5: DLC2 and Kif1B are required for mitotic checkpoint satisfaction.(a) HeLa cells expressing H2B-mCherry and α-tubulin-GFP were transfected with control, DLC2 or Kif1B siRNAs and filmed through mitosis. Pictures were taken with a Spinning Disc confocal microscope every 2 min (see Supplementary Movies 1–3). (b) Length of prometaphase and metaphase were quantified (Box-and-whisker plots, n=30). (c) Examples of control, DLC2- and Kif1B siRNA-transfected HCE cells stained for CREST (green) and Mad2 (red). (d) Quantification of the percentage of cells showing a specific number of Mad2-positive dots at the kinetochores. DLC2 and Kif1B KD cells show higher percentages of cells with more than one Mad2-positive dot (shown are means±1 s.d.; n=3 experiments). Scale bars, 10 μm. (*P<0.05, ***P<0.001; analysis of variance).