Cdh1 and AurB kinase influence cell spreading in early G1. (A,B) U2OS-AurB–Venus cells were treated with the indicated drugs after anaphase onset (doses in Materials and Methods) and recorded by time-lapse microscopy as in Fig. 1F. (A) Results from multiple experiments plotted as bars representing trajectories of individual cells. A statistical summary of these data can be found in Table 1. (B) Frames from representative movies with times adjusted to anaphase onset. Drug treatments occurred at the following times after anaphase for the cells shown: MG132 (13 minutes), nocodazole (noco; 8 minutes), taxol (10 minutes), cytochalasin D (cytoD; 8 minutes). Brightness and contrast have been adjusted in the enlarged images of the boxed regions to show AurB accumulation (or lack thereof) at the edge of the cell. Arrows indicate midbodies. (C) Representative frames from fluorescence time-lapse microscopy of non-confluent cell cultures of RPE-α-actinin–Venus, with the appearance of α-actinin-associated structures scored by arrows (actin fibres) or arrowheads (leading edges). The images on the right are enlargements of the 46 minutes time point. See supplementary material Movies 4–6. Drugs, as indicated, were added at the following times after anaphase onset: MG132, 8 minutes; ZM, 10 minutes; Cdh1i/ZM, 17 minutes. (D) The timings of actin fibre assembly using time-lapse data collected during two or more experiments and plotted as means ± s.d. P-values were calculated using Student's t-test. (E) Time-lapse movies of RPE-α-actinin–Venus cells were used to measure the area of daughter cells during cell spreading (upper panel). Length of daughter cells was measured from midbody to cell edge along the axis defined by the position of the anaphase spindle and a cell shape index calculated by expressing the cell area as a function of cell length (lower panel). Distribution of measurements from individual cells are shown: elongated, polarized daughter cells give the lowest cell shape index. *P<0.01, **P<0.001, Student's t-test. Scale bars: 20 µm.

FHOD1 controls AurB localisation and cell spreading during return to interphase. (A) AurB–Venus-expressing cells were transfected with HA-tagged FHOD1, fixed and stained after 24 hours with antibodies against FHOD1 and GFP (left-hand panel) or HA and AurB (middle panel). Other cells were treated briefly with MG132 (as in Fig. 1A) and stained for pAur and HA (right-hand panel). Arrows indicate some sites of colocalisation. Scale bars: 10 µm. (B) Individual cell trajectories from populations of AurB–Venus cells treated with control or FHOD1 siRNA (supplementary material Movies 7, 8). See also supplementary material Fig. S3. (C) Frames from representative movies of RPE-α-actinin–Venus cells filmed as in Fig. 2C after treatment with control or FHOD1 siRNA. Scale bars: 20 µm. (D) Time-lapse movies of FHOD1-i RPE-α-actinin–Venus cells were used to calculate cell shape indices as described for Fig. 2E. Gl2-i and Cdh1-i results from Fig. 2E are included for comparison. (E–I) U2OS cells synchronized and fixed to optimize staining for F-actin (using phalloidin), for MTs (β-tubulin) or for MT +TIPS (EB3). Cells were treated with control or FHOD1 siRNA, or FHOD1-i together with siRNA-resistant versions of HA-FHOD1 where indicated. Representative images are shown in E, G and I. (F) Values for relative F-actin staining at the cortex in individual cells were calculated as the ratio of maximum value at the cell periphery to average pixel value across the cell (as described in more detail in legend to Fig. 4E) and are plotted here against the circularity measurement for each cell, measured as described in legend to Fig. 3H. Trend line indicates the inverse relationship between peripheral F-actin staining and cell shape. (H) Cell shape measurements were made from images of 40 or more cells acquired from two separate experiments. Circularity is measured on a scale of 0–1, where 1 represents a perfect circle (see Materials and Methods for details). ***P<0.0001 by Student's t-test. (I) Representative images of cells stained for EB3. ZM was added 15 minutes prior to fixation, where indicated. Images of the boxed regions show regions of the cell edge under increased magnification and contrast, all pixels were treated in an identical fashion. See also supplementary material Fig. S3. Scale bars: 5 µm.

FHOD1 phosphorylation by AurB coordinates F-actin assembly. (A) Schematic of FHOD1 domain organization showing known functional domains and AurB phosphorylation sites identified by mass spectrometry. Further details are in supplementary material Fig. S4. S/T residues in bold were mutated to generate FHOD1-5D and −5A. GBD, GTPase binding domain; DAD, Dia autoregulatory domain; FH, formin homology. (B) In vitro kinase assay comparing HA–FHOD1wt with HA–FHOD1-5A after immuno-isolation from HEK293T cells and in vitro kinase reaction with recombinant AurB. The upper panel depicts an autoradiograph of the kinase reaction, the lower panel an immunoblot to illustrate the amounts of FHOD1 protein in each kinase reaction. (C) Quantification of the FHOD1 phosphorylation shown in B. Depicted is the ratio of the phospho-FHOD1 relative to total FHOD1 protein signal, normalized to the values obtained for FHOD1wt (mean of three independent experiments ± s.d.). (D–G) U2OS cells were prepared by FHOD1 siRNA-rescue to express different versions of HA–FHOD1, synchronized in early G1 phase and fixed with PFA. (D) Representative images of cells examined for the presence of F-actin, MTs and HA. Arrows indicate prominent F-actin cables at the periphery of daughter cells. Arrowheads in enlarged images indicate sites of colocalisation of labels. Scale bars: 20 µm. (E) F-actin staining was profiled along a cell section (indicated by yellow arrow, left-hand panel) bisecting the direction of maximum spread of daughter cells. Relative staining at the cortex was calculated as the ratio of the maximum value at the cell periphery to average pixel value across the cell. The distribution of data collected from more than 40 cells from two experiments are shown as box-plots. ***P<0.001, *P<0.01 by Student's t-test. (F) Cells from the same experiments were scored for colocalisation of HA–FHOD1 with MTs or SFs at the cell cortex. Bar chart shows percentage of the total number of HA-positive cells examined in different categories. (G) Cell shape analysis was carried out as in Fig. 3G. **P<0.001, *P<0.01 by Student's t-test.