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Cdk1-dependent mitotic enrichment of cortical myosin II promotes cell rounding against confinement

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Abstract

Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (<10 s) resistance to mechanical deformation, myosin sustains intracellular pressure for a longer duration (>60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.

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Figure 1: F-actin and myosin II are enriched at the cortex during mitosis.
Figure 2: Transmitotic localization of cortical myosin II coincides with buildup of intracellular pressure.
Figure 3: Enrichment of cortical F-actin is independent of myosin II activity.
Figure 4: Cdk1 activity is essential for the enrichment of cortical myosin II and elevated intracellular pressure.
Figure 5: Imposed confinement of rounded cells yields a characteristic time-dependent mechanical response.
Figure 6: Myosin II-inhibited mitotic cells are unable to sustain persistent pressure.
Figure 7: Localization of myosin II depends on Rho kinase and F-actin scaffolding, which is sustained by formin-mediated nucleation.
Figure 8: Modulation of transmitotic intracellular pressure and cortical tension through regulation of the actomyosin cortex.

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Acknowledgements

We thank S. Weiser, Y. Toyoda, S. Raghuraman, R. Newton and B. Sorce for input, S. Hayashi, T. Kondo, M. Piel, C. Cabernard and O. Pertz for critical reading of the manuscript, the E. Paluch and F. Buchholz laboratories for contributing HeLa cell lines, J. Mercer for providing the mutant Rho GTPase plasmids as well as Cdc42 inhibitor, pirl1, and T. Horn and A. Ponti for help with the experiments on fluorescence recovery after photobleaching. This work was supported by the Eidgenössische Technische Hochschule Zürich (ETH research grant ETH-05 11-2), Swiss National Science Foundation (advanced mobility fellowship to M.P.S. and grant 31003A_138063 to D.J.M.) and National Center of Competence in Research Nano.

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Authors

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S.P.R., J.H., M.P.S., A.A.H. and D.J.M. planned the research and wrote the paper. S.P.R. designed, carried out and analysed most experiments. M.P.S. designed the STC-arrest methodology. M.P.S. and C.J.C. contributed to experiments in Supplementary Figs 7 and 8 respectively. J.H. wrote the scripts for image analysis.

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Correspondence to Anthony A. Hyman or Daniel J. Muller.

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Integrated supplementary information

Supplementary Figure 3 Quantification of cortex/cytoplasm ratio and cortical full width half maximum (FWHM) of labeled F-actin and myosin II in live cells.

(a) Confocal images of a MYH9-GFP Lifeact-mCherry expressing HeLa cell were analyzed with a self-written Igor macro (Igor Pro). The center of the cell (purple circle) was determined by fitting a circle to the edge of the MYH9-GFP signal and 60 points (yellow markers) were initially positioned 6° apart along the circle. Next, the 60 points were automatically repositioned to be at the cell periphery. (b) Fluorescence intensity profile (cyan) taken from the average signal along a 0.6 μm thick radial line (yellow) through a representative peripheral point (yellow marker). (c) The 60 fluorescence intensity profiles (thin traces) through the peripheral yellow markers were aligned at the cell periphery (0 μm) and superimposed. The thick green trace represents the average of all 60 fluorescent intensity profiles taken from one cell. (d) Determination of cortex/cytoplasm ratio (R) of the whole cell from the average fluorescence intensity profile. Within each average profile, the peak intensity (p) between −2 and 0.5 μm from the cell edge and the mean intensity (m) between −2 and −1 μm from the cell edge were extracted. The cortex/cytoplasm ratio (R) for the fluorescence image was determined as the ratio of p and m. (e) Determining the cortical FWHM of the average fluorescence intensity profile shown in (d). The half peak height (I) is the intensity halfway between p and m. Cortical FWHM is the width (in μm) of the signal at I. (f) The yellow markers from the MYH9-GFP image were copied into the Lifeact-mCherry fluorescence image. (g) Fluorescence intensity profile (cyan) taken from the average signal along a 0.6 μm thick radial line (yellow) through a representative peripheral point (yellow marker). (h) The 60 fluorescence intensity profiles (thin traces) through the peripheral yellow markers were superimposed and aligned at the cell periphery (0 μm). The thick green trace represents the average of all intensity profiles. (i) Determination of cortex/cytoplasm ratio (R) of the whole cell from average fluorescence intensity profile. Within each average profile, the peak intensity (p) between −2 and 0.5 μm from the cell edge and the mean intensity (m) between −2 and −1 μm from the cell edge were extracted. The cortex/cytoplasm ratio (R) for the fluorescence image was determined as the ratio of p and m. (j) Determining the cortical FWHM from the average fluorescence intensity profiles of a whole cell. The half peak height (I) is the intensity halfway between p and m. Cortical FWHM is the width (in μm) of the signal at I. (k) Superimpositions of the average F-actin (red) and myosin II (green) fluorescence intensity profiles, from c and h, of a mitotic cell. (i) Average intensity profile quantifying the radial distribution of F-actin (red) and myosin II (green) in a mitotic cell treated with 20 μg ml−1 C3-toxin.

Supplementary Figure 4 Enrichment and turnover of cortical actin during mitosis is not dependent on myosin II.

(a) Images showing a cross sectional region of an STC-arrested mitotic β-actin-GFP expressing HeLa cell used in a photobleaching experiment. The region inside the red circle (diameter, 2 μm) was photobleached for three seconds before time zero. The actin cortex/cytoplasm ratio of the unbleached cell cortex is the ratio of mean pixel intensity in the yellow square (0.32 μm2), outside the bleach zone, and the magenta rectangle (0.32 × 1.18 μm2), which is 0.32 μm away from the unbleached region of the cortex in the cytosol. The actin cortex/cytoplasm ratio of the bleached cell cortex is the ratio of mean pixel intensity in the green square (0.32 μm2), within the bleach zone, and the magenta rectangle. Scale bar, 1.25 μm. (b) The actin cortex/cytoplasm ratio for unbleached (black) and bleached (magenta) cell cortex. The blue trace is the exponential fit of the data, post-bleaching. The half-time and recovery for the cell are indicated within the plot. (c) Plot of the time taken after photobleaching to reach half the final actin cortex/cytoplasm ratio (Half-time) and the percentage recovery of actin cortex/cytoplasm ratio compared to pre-bleached cortex. Cells were transfected at least 48 hours prior to the experiment with control (n = 9 cells) or MYH9 (n = 9 cells) siRNA. The diamond-box contains 25–75% percentiles of the data and the bar denotes the median. n indicates the number of single-cell experiments. Mann–Whitney significance tests of cells with respect to data plotted in the first column are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005.

Supplementary Figure 5 Cdk1 inhibition in the presence of mitotic spindle triggers anisotropic localization of cortical F-actin and myosin II.

(a) Confocal images of H2B-mCherry (red) and tubulin-GFP (green) expressing mitotic HeLa cells. The cells were either untreated or treated with the indicated inhibitor to induce mitotic arrest. Protease inhibitor, MG132, treated cells exhibited bipolar morphology similar to untreated cells. STC, an Eg5 inhibitor, treated cells had mono-polar spindle morphology. Microtubule depolymerizer, nocodazole (Noc), treated cells were devoid of mitotic spindle. Scale bar of 10 μm applies to all images. (b) Confocal images of MYH9-GFP (green) and Lifeact-mCherry (red) expressing mitotic HeLa cells. Images were acquired every 4 minutes. Cells were untreated, or preincubated for 30 min with MG132, STC or nocodazole (Noc) at concentrations indicated. Nine minutes after starting the experiment 3 μM Cdk1/2 inhibitor III was introduced. Scale bar of 10 μm applies to all images. (c) Scheme of the mitotic spindle organizing the cortical enrichment of actomyosin when Cdk1 is inhibited. In Cdk1 inhibited cells with a bi (untreated or MG132 arrest) or mono (STC arrest) polar spindle, cortical actomyosin is enriched in regions furthest from the spindle poles. Mitotic cells with no spindle (nocodazole arrest) were devoid of cortical myosin II enrichment upon Cdk1 inhibition.

Supplementary Figure 6 Increasing cell confinement does not increase cortex tension and intracellular pressure in mitotic cells.

(a) Plot of cell radius and volume of STC arrested mitotic MYH9-GFP Lifeact mCherry expressing HeLa cells subjected to imposed confinement. The cells were confined to heights indicated (16 μm, n = 15 cells; 14 μm, n = 11 cells; 12 μm, n = 14 cells; 10 μm, n = 13 cells; 8 μm, n = 11 cells) and measured 60 seconds after confinement. (b) Quantification of cortex/cytoplasm ratio and cortical FWHM of F-actin (red) or myosin II (green). The cells were confined to heights indicated and measured 60 seconds after confinement. (c) Quantification of transient and persistent force (purple), pressure (grey) or tension (blue) of cells. The results show that with increased confinement the intracellular pressure and cortical tension decrease rather than increase. This decrease could be due to the increase in the surface area of the cell cortex upon confinement. Increase in cortex surface area would reduce the density of myosin motors in the cortex, which in turn could reduce cortex tension and intracellular pressure. The diamond-box contains 25–75% percentiles of the data and the bar denotes the median. Mann–Whitney significance tests of mitotic cells with respect to data plotted in the first column are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005.

Supplementary Figure 7 Myosin IIA depleted mitotic cells are compromised in sustaining persistent pressure.

(a) Representative confocal images of STC-arrested mitotic MYH9-GFPLifeact-mCherry expressing HeLa cells confined to a height of 10 μm. Cells were transfected at least 48 hours prior to experiments with control (n = 9 cells), Ect2 (n = 13 cells) or MYH9MYH9 (n = 10 cells) siRNA. Scale bars, 10 μm. (b) Cortex/cytoplasm ratios of F-actin (red) and myosin II (green). Cells were transfected at least 48 hours prior to experiments with control (n = 9 cells), Ect2 (n = 13 cells) or MYH9 (n = 10 cells) siRNA. (c) Average force traces of STC-arrested mitotic MYH9-GFP Lifeact-mCherry expressing HeLa cells confined to a height of 10 μm. Time zero represents arrival at the height of 10 μm after the AFM-cantilever descended at a speed of 1 μm s−1. Cells were transfected with control (orange, n = 9 cells), Ect2 (red, n = 13 cells) or MYH9 (purple, n = 10 cells) RNAi. (d) Transient or persistent pressure (grey) and tension (blue) of mitotic cells under confinement. Cells were transfected at least 48 hours prior to experiments with control (n = 9 cells), Ect2 (n = 13 cells) or MYH9 (n = 10 cells) siRNA. The diamond-box contains 25–75% percentiles of the data and the bar denotes the median. Mann–Whitney significance tests of mitotic cells with respect to data plotted in the first column are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005.

Supplementary Figure 8 Unlike RhoA, inhibition of Rac1 or Cdc42 does not affect mitotic cortex tension or intracellular pressure.

(a) Plotted are persistent pressure (grey) and tension (blue) of STC-arrested mitotic HeLa cells that were untreated (n = 84 cells) or incubated with C3-toxin (n = 11, 8 and 18 cells), NSC23766 (NSC, n = 47 and 12 cells), EHT1864 (EHT, n = 37 and 15 cells), Pirl1 (Pirl, n = 46 and 32 cells) or ML141 (n = 35 and 22 cells) at concentrations given. Mann–Whitney significance tests of mitotic cells with respect to data plotted in the untreated cells are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005. (b) Activation of RhoA or inhibition of PAK1-3 increases mitotic blebbing. Plots characterizing STC-arrested mitotic HeLa cells expressing Lifeact-mCherry and MYH9-GFP that were untreated (n = 82) or pre-incubated for at least 30 minutes with latrunculin A (LatA, n = 10 and 7 cells), blebbistatin (Blebb, n = 10 and 10 cells), calpeptin (Calp, n = 10 and 9 cells), C3-toxin (C3, n = 7 and 7 cells), Y27632 (Y27, n = 10 and 10 cells), SMIFH2 (SMI, n = 11 and 10 cells), CK666 (CK, n = 10 and 10 cells) or IPA3 (n = 9 and 10 cells) at the concentrations given (method as in Fig. 5). Bars (grey) indicate the percentage of round mitotic cells that blebbed when confined to 10 μm. Number of cells tested is noted above each bar. The red and green plots indicate the cortical FWHM of F-actin and myosin II, respectively. In cases where there was no enrichment of F-actin or myosin II at the cortex, cortical FWHM has not been shown. The diamond-box contains 25–75% percentiles of the data and the bar denotes the median. Mann–Whitney significance tests of mitotic cells with respect to data plotted in the first column are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005.

Supplementary Figure 9 PAK1-3 inactivated mitotic cells exhibit accelerated intracellular pressure buildup in early mitosis.

(a) Representative plots of the pressure of two MYH9-GFP H2B-mCherry expressing HeLa cell. The cells measured were untreated (orange) or pre-incubated for at least 3 hours with 20 μM IPA3 (purple). The measurements started at NEBD (defined as time zero). Fluorescence time-lapse images of the cells are included at the top. Scale bar, 20 μm, applies to all images. (b) The maximum pressure attained during mitotic progression (max pressure) and the time taken from NEBD until a pressure of 300 Pa was reached (time to 300 Pa) by MYH9-GFP H2B-mCherry expressing HeLa cell. Cells were untreated (n = 8 cells) or pre-incubated for at least 3 hours with 20 μM IPA3 (n = 5 cells). (c) Scheme to probe the opposing roles of Rho kinase and PAKs in regulating cortical myosin II enrichment. STC-arrested mitotic MYH9-GFP and Lifeact-mCherry expressing HeLa cells were treated with Rho kinase and PAK1-3 inhibitors simultaneously while the enrichment of cortical myosin II and F-actin were tracked. (d) Myosin II and F-actin cortex/cytoplasm ratio of mitotic cells. The data plotted are taken eight minutes prior to (control, n = 71 cells) or 20 minutes after the introduction of 60 μM IPA3 and Y27632 (0 μM, n = 12; 0.1 μM, n = 8; 0.5 μM, n = 15; 1.25 μM, n = 11; 2.5 μM, n = 15 cells,) at the indicated concentration. (e) Myosin II and F-actin cortex/cytoplasm ratio of mitotic cells. The data plotted are eight minutes prior to (control, n = 50 cells) or 20 minutes after the introduction of 10 μM FRAX486 and Y27632 (0 μM, n = 8; 0.1 μM, n = 7; 0.5 μM, n = 10; 1.25 μM, n = 8; 2.5 μM, n = 10 cells) at the indicated concentration. The diamond-box contains 25–75% percentiles of the data and the bar denotes the median. Mann–Whitney significance tests of mitotic cells with respect to data plotted in the first column are indicated: (NS) U > 0.05, () 0.05 ≥ U > 0.005, (U ≤ 0.005.

Supplementary Figure 10 DIAPH1 depleted mitotic cells are compromised in maintaining cortical F-actin enrichment.

(a) Representative confocal images of STC-arrested mitotic MYH9-GFP and Lifeact-mCherry expressing HeLa cells confined to a height of 10 μm. Cells were transfected at least 48 hours prior to experiments with control, MYH9, DIAPH1_1 (C1) or DIAPH1_2 (C2) siRNA. Both C1 and C2 target the same DIAPH1 gene. Scale bars, 10 μm. (b) Cortex/cytoplasm ratios of F-actin (red) and myosin II (green). Cells were transfected at least 48 hours prior to experiments with control (n = 10 cells), MYH9 (n = 10 cells), DIAPH1_1 (C1, n = 10 cells) or DIAPH1_2 (C2, n = 8 cells) siRNA. (c) Transient or persistent pressure (grey) and tension (blue) of mitotic cells under confinement. (d) Assay to establish that CK666 inhibits Arp2/3 in mitotic cells. In mitotic cells, dynamic clusters of F-actin revolve with the cytosol with a regular frequency. This phenomenon has been shown to be Arp2/3 dependent (Mitsushima, M. et al., 2010). Shown are confocal images of Lifeact-mCherry to visualize amorphous cytosolic F-actin structures in STC-arrested mitotic MYH9-GFP and Lifeact-mCherry expressing HeLa cells. Images were acquired every 2 minutes. The acquisition time is indicated at the top left corner. At time zero, 40 μM CK666 was introduced to previously untreated medium. An asterisk indicates the presence of amorphous cytosolic F-actin structures in the vicinity. Within 20 minutes upon CK666 introduction, the cytosolic F-actin clusters start to disappear. Scale bar of 10 μm applies to all images.

Supplementary Table 1 Mitotic localizations of actin and myosin reported in the literature.
Supplementary Table 2 Inhibitors and perturbations used in single cell experiments in order of figure appearance.

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Ramanathan, S., Helenius, J., Stewart, M. et al. Cdk1-dependent mitotic enrichment of cortical myosin II promotes cell rounding against confinement. Nat Cell Biol 17, 148–159 (2015). https://doi.org/10.1038/ncb3098

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