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. 2020 Apr 7;31(1):107477.
doi: 10.1016/j.celrep.2020.03.041.

Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis

Nilay Taneja  1 , Matthew R Bersi  2 , Sophie M Baillargeon  1 , Aidan M Fenix  1 , James A Cooper  1 , Ryoma Ohi  3 , Vivian Gama  1 , W David Merryman  2 , Dylan T Burnette  4
Affiliations

Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis

Nilay Taneja et al. Cell Rep. .

Abstract

The mechanical properties of the actin cortex regulate shape changes during cell division, cell migration, and tissue morphogenesis. We show that modulation of myosin II (MII) filament composition allows tuning of surface tension at the cortex to maintain cell shape during cytokinesis. Our results reveal that MIIA generates cortex tension, while MIIB acts as a stabilizing motor and its inclusion in MII hetero-filaments reduces cortex tension. Tension generation by MIIA drives faster cleavage furrow ingression and bleb formation. We also show distinct roles for the motor and tail domains of MIIB in maintaining cytokinetic fidelity. Maintenance of cortical stability by the motor domain of MIIB safeguards against shape instability-induced chromosome missegregation, while its tail domain mediates cortical localization at the terminal stages of cytokinesis to mediate cell abscission. Because most non-muscle contractile systems are cortical, this tuning mechanism will likely be applicable to numerous processes driven by myosin-II contractility.

Keywords: actin cortex; binucleation; bleb; cell division; cortex tension; cytokinesis; hydrostatic pressure; myosin IIA; myosin IIB; spindle.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. MIIA Drives Furrow Ingression through Templating of MII Stacks
(A and B) Endogenous MIIB (A) and MIIA (B) in the cleavage furrow of Scr versus MIIAlo or MIIBlo cells, respectively. (Insets) Zoom of yellow box. For (A), n = 13 Scr cells, N = 4 experiments; n = 17 MIIAlo cells, N = 5 experiments. For (B), n = 14 Scr and 8 MIIBlo cells, N = 3 experiments. Schematic, labeling strategy. (C)Average length of MII stacks. (D) Furrow ingression rate from phase contrast at 20× magnification. (E) Tukey plots of ingression from phase contrast. n = 18 Scr, 22 MIIAlo (pooled siRNA) and 20 MIIBlo (pooled siRNA) cells, N = 3 experiments. (F) Tukey plots of ingression measured independently using DIC at 60× magnification. n = 18 Scr, 16 MIIAlo (single siRNA), and 14 MIIAlo+MIIA cells, N = 4 experiments. (Note: phase contrast systematically overestimates distance measurements and gives higher values of ingression. Hence, the y axis is scaled differently for E.) (G) Tukey plots of ingression measured independently using DIC. n = 15 MIIAlo (pooled siRNA) and 15 MIIAlo+MIIB cells, N = 3 experiments. The Scr dataset is the same as (F) and is only displayed for comparison. (H) Representative kymographs of parental, MIIA-KO, MIIA-KO+MIIA-mEGFP, and MIIA-KO+MIIB-mEGFP HAP1 cells. (I) Tukey plots of ingression for parental, MIIBlo (single siRNA), MIIA-KO, MIIA-KO+MIIA-mEGFP, or MIIA-KO+MIIB-mEGFP HAP1 fibroblasts. n = 11 MIIA KO, 12 MIIA-expressing, and 10 MIIB-expressing cells, N = 3 experiments. Solid circles represent outliers. (Inset) Western blot showing siRNA knockdown of MIIB in parental HAP1 fibroblasts with tubulin as loading control. Error bars in (C) represent standard error of the means. Scale bar, 5 mm. p values stated over graphs.
Figure 2.
Figure 2.. MII Paralog Depletion Leads to Distinct Alterations to Cell Shape
(A) Early, mid, and late cytokinesis in Scr, MIIBlo, MIIAlo, MIIBlo+ MIIB-mEGFP, and MIIBlo+ MIIA-mEGFP cells. Yellow arrows denote blebs. (B and C) Tukey plots of blebbing measured as sum diameter of blebs per minute. (B) n = 18 Scr, 15 MIIBlo (pooled siRNA), 16 MIIBlo (single siRNA), 15 MIIBlo +MIIB, and 10 MIIBlo +MIIA cells, N = 4 experiments. (C) n = 14 MIIAlo (pooled siRNA), 14 MIIAlo (single siRNA), 14 MIIAlo +MIIA, and 15 MIIAlo +MIIB cells; N = 4 experiments. (D) Control M2 cells 5-h post plating. (Insets) Examples of not-blebbing (1) versus blebbing (2) cells. (E) Knockdown of MIIA and MIIB in M2 cells. Intensity of MII bands normalized to tubulin loading control shown below each band. (F) Proportion of blebbing cells in Scr, MIIAlo, MIIBlo, or MIIAlo+MIIA-mEGFP. Number of cells stated inside bars. (G) Scr, MIIAlo, and MIIBlo cells at metaphase and late cytokinesis. (H) Kymographs were generated using dotted white lines in (G). Dotted yellow lines show the two pole-to-pole lengths used to measure elongation ratio. SeeMethod Details for quantification details. (I) Tukey plots of polar elongation. n = 17 Scr, 20 MIIAlo (pooled siRNA) and 18 MIIBlo (pooled siRNA) cells, N = 3 experiments. Scale bars, (A) and (G) 10 μm; (D) 100 μm; (inset) 25 μm. p values stated over graphs.
Figure 3.
Figure 3.. MII Paralog Compensation at the Polar Cortex upon MII Knockdown Results in Altered Hetero-Filament Composition
(A and B) Early, mid, and late cytokinesis cells showing endogenous MIIA (A) or MIIB (B) (Fire LUT) in Scr versus MIIBlo or MIIAlo cells, respectively. All compared images were scaled similarly. (C) Intensity of MIIA and MIIB at the polar cortex calculated as mean of green regions of interest (ROIs) in cartoon insets. For MIIA: n = 41 Scr and 48 MIIBlo cells, N = 3 experiments. For MIIB: n = 46 Scr and 43 MIIAlo cells, N = 3 experiments. (D) MIIA and MIIB localization (Fire LUT) during metaphase in MIIBlo and MIIAlo cells, respectively. (E) MIIA: n = 15 cells each for Scr and MIIBlo, N = 3 experiments. MIIB: n = 13 Scr cells and 12 MIIAlo cells, N = 3 experiments. (F) MIIA and MIIB localization (mpl inferno LUT for single channel) in control versus knockdown cells using SIM. (G) Tukey plots showing percentage of MIIA filaments that co-localized with MIIB. Solid circles represent outliers. (H) Mean intensity of MIIA and MIIB upon knockdown. n = 503, 361, and 354 filaments in Scr, MIIAlo and MIIBlo cells, respectively, from 10 cells per condition; N = 3 experiments. Error bars in (C) and (E) represent standard error of weighted mean. Error bars in (H) represent standard error of the means. Scale bars, (A), (B), and (D) 10 μm; (F) 1 μm. p values stated over graphs.
Figure 4.
Figure 4.. MIIA Is Necessary and Sufficient to Generate Cortex Tension and Intracellular Pressure
(A) Micropipette aspiration of control cell during metaphase, showing cell radius (Rc), aspirated length (L), and radius of pipette (Rp). Tension was calculated using the mathematical expression shown. (B) Tukey plots of cortex tension upon MII paralog depletion. n = 16 Scr, 14 MIIAlo, 15 MIIBlo, 10 MIIAlo +MIIA-mEGFP, 11 blebbistatin-treated, and 12 MIIBlo +MIIB-mEGFP cells, N = 4 experiments each. (C) Scr, MIIAlo, MIIBlo, and 50 μM of blebbistatin-treated HeLa cells before ablation, 0 s after ablation, and 90 s after ablation of the metaphase cortex. Yellow squares depict ablation ROI, yellow arrows depict blebs. (D) Tukey plots showing initial bleb size during metaphase in HeLa cells. n = 17 Scr, 16 MIIAlo, 14 MIIAlo +MIIA, 14 50-mM blebbistatin-treated, and 18 MIIBlo cells, N = 3 experiments. (E) Polar cortex ablation in Scr, MIIAlo, and MIIBlo HeLa cells during cytokinesis. Yellow squares depict ablation ROI, yellow arrows denote bleb created by ablation. (F) Tukey plots of initial bleb size during cytokinesis in HeLa cells. n = 25 Scr, 15 MIIAlo, and 25 MIIBlo cells, N = 3 experiments. (G) Tukey plots of initial bleb size following ablation in HAP1 KO fibroblasts during cytokinesis. n = 12 untransfected MIIA-KO-, 26 MIIA-, and 11 MIIB-expressingcells, N = 3 experiments. Scale bars, 10 mm. Solid circles represent outliers. p values stated over graphs.
Figure 5.
Figure 5.. Motor Domains Determine the Contribution of MII Paralogs to Cortical Contractility
(A) Localization of MIIB/A-mEGFP and MIIA/B-mEGFP chimera in MIIBlo and MIIAlo cells, respectively. Arrow indicates enrichment of MIIA/B at the equatorial cortex during late cytokinesis. (B) MIIBlo cell expressing MIIB/A chimera at metaphase and cytokinesis. Tukey plots of blebbing events in MIIB/A-expressing cells compared to Scr and MIIBlo (single siRNA). n = 16 MIIBlo +MIIB/A cells, N = 3 experiments. The Scr and MIIBlo (single) datasets are the same as in Figure 2B and are only shown for comparison. (C) MIIAlo cell expressing MIIA/B chimera at metaphase and cytokinesis. n = 16 MIIAlo +MIIA/B cells, N = 3 experiments. The Scr and MIIAlo (single) datasets are the same as Figure 2C and are only shown for comparison. (D) Cleavage furrow ingression rates using DIC comparing MIIAlo cells expressing MIIA/B chimera. n = 16 MIIAlo +MIIA/B cells, N = 3 experiments. The Scr and MIIAlo (single) datasets are the same as Figure 1F and are only shown for comparison. (E) Pole-to-pole elongation ratios for MIIAlo cells expressing MIIA/B chimera. n = 12 MIIAlo +MIIA/B cells, N = 3 experiments. The Scr and MIIAlo (single) datasets are the same as Figure S3 and are only shown for comparison. (F) Cortex tension in MIIB/A and MIIA/B chimeras using micropipette aspiration. n = 14 MIIAlo +MIIA/B- and 10 MIIBlo +MIIB/A-expressing cells, N = 4 experiments. The Scr, MIIAlo, and MIIBlo datasets are the same as in Figure 4B and are only shown for comparison. (G) Initial bleb size following ablation in HAP1 KO cells. n = 8 MIIB/A- and 18 MIIA/B-expressing cells, N = 3 experiments. Yellow and white arrows denote formation or absence of a bleb, respectively. The control KO dataset is the same as Figure 4G and is only shown for comparison. Scale bars, (A and G) 10 μm; (B and C), 5 μm. Solid circles represent outliers. p values stated over graphs.
Figure 6.
Figure 6.. MIIB Depletion Leads to Increased Binucleation
(A) Scr, MIIAlo, and MIIBlo cells 48 h post re-plating, showing F-actin (green) and nuclei (magenta). Yellow arrows denote binucleated cells. (Inset) Higher magnification examples, 1–4. (B) Binucleate cells 48-h post plating in Scr versus MIIAlo (pooled siRNA) or MIIBlo (pooled siRNA) cells. n > = 1,000 cells each, N = 3 experiments. (C) Binucleate cells 48-h post plating in MIIBlo (single siRNA) versus MIIBlo cells expressing MIIB, MIIB/A, or MIIA/B. n = 760 MIIBlo (single), 584 MIIB-expressing, 464 MIIB/A-expressing, and 347 MIIA/B-expressing MIIBlo cells, N = 3 experiments. (D) Representative binucleated MIIBlo cells adjacent to enucleated cells. (E) Binucleated cells adjacent to an enucleated cell, as a fraction of total number of cells. Scale bars, (A) 50 μm; (inset), 25 μm; (D) 25 μm. Error bars represent standard error of the mean. p values stated over graphs.
Figure 7.
Figure 7.. Loss of MIIB Drives Binucleation through Two Distinct Mechanisms
(A) Time montage showing displacement of the chromosomes (dotted yellow lines) with the retraction of a large bleb (yellow arrow) in a MIIBlo cell. (B) Correlation of spindle displacement with the size and timing of blebbing events. Each data point represents a bleb/displacement event plotted over 3 experiments. (C) Comparison of oscillating versus non-oscillating cells with respect to bleb size and number of blebbing events. Each data point represents a cell. n = 36 cells,N = 6 experiments. (D) Representative time montage of spindle oscillation-induced chromosome missegregation upon MIIB depletion. (E) Representative time montage showing abscission failure upon MIIB depletion. See Figure S7B for extended time montage. The ROI used to create the montage was moved to keep the cells centered in the field of view. (F) Relative proportions of the modes of binucleation upon MIIB depletion. n = 40 MIIBlo, 11 MIIBlo+MIIB/A, and 14 MIIBlo+MIIA/B binucleation events. Error bars in (C) represent standard deviation. Scale bars, 10 μm (A); 20 μm (D); 50 μm (E). p values stated over graphs.
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References

    1. Beach JR, Shao L, Remmert K, Li D, Betzig E, and Hammer JA 3rd. (2014). Nonmuscle myosin II isoforms coassemble in living cells. Curr. Biol 24, 1160–1166. - PMC - PubMed
    1. Bekker-Jensen DB, Kelstrup CD, Batth TS, Larsen SC, Haldrup C, Bramsen JB, Sorensen KD, Hoyer S, Orntoft TF, Andersen CL, et al. (2017). An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst. 4, 587–599 e584. - PMC - PubMed
    1. Bement WM, Leda M, Moe AM, Kita AM, Larson ME, Golding AE, Pfeuti C, Su KC, Miller AL, Goryachev AB, and von Dassow G (2015). Activator-inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium. Nat. Cell Biol 17, 1471–1483. - PMC - PubMed
    1. Bovellan M, Romeo Y, Biro M, Boden A, Chugh P, Yonis A, Vaghela M, Fritzsche M, Moulding D, Thorogate R, et al. (2014). Cellular control of cortical actin nucleation. Curr. Biol 24, 1628–1635. - PMC - PubMed
    1. Brugués J, Maugis B, Casademunt J, Nassoy P, Amblard F, and Sens P (2010). Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration. Proc. Natl. Acad. Sci. USA 107, 15415–15420. - PMC - PubMed

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