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, 160 (2), 255-65

RhoA Is Required for Cortical Retraction and Rigidity During Mitotic Cell Rounding

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RhoA Is Required for Cortical Retraction and Rigidity During Mitotic Cell Rounding

Amy Shaub Maddox et al. J Cell Biol.

Abstract

Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.

Figures

Figure 1.
Figure 1.
RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. HeLa cells growing on glass coverslips were treated with recombinant GST–C3 toxin or GST alone (0.28 μg per 24-well plate well) by introduction with LipofectAmine reagent for 90 min. Cells were fixed and stained for F-actin, paxillin, and DNA. Cells in metaphase of mitosis were identified as having a metaphase plate of aligned chromosomes. For metaphase cells, diameter, perimeter, and area were measured by drawing on the phase image (C, D, Q, and R). The diameter measurement reported is the cell diameter (black line) bisecting and perpendicular to the diameter that passes through the metaphase plate (gray line). GST-treated metaphase cells are well rounded, whereas GST–C3-treated metaphase cells are not completely rounded (compare C with D). Bar, 15 μm. (S) Quantitation of measurements of mitotic cells. Measurements are averages and standard deviations from one of three equivalent experiments. n ≥ 21 for each experiment. *, significant difference from GST (P < 0.0005).
Figure 2.
Figure 2.
Rho-kinase is required for cortical retraction during mitotic cell rounding. HeLa cells were treated with Y-27632 or vehicle alone (Control) in growth medium for 1 h. Cells were fixed and stained for F-actin and DNA. Phase contrast and fluorescence images are shown. Bar, 15 μm. (G) Cortical retraction was quantified as for Fig. 1. Measurements are averages and standard deviations from one of three equivalent experiments. n ≥ 30 for each experiment. *, significant difference from Control (P < 0.0005).
Figure 3.
Figure 3.
RhoA is required for rigidity of rounded mitotic cells. (A) Phase images of HeLa cells and the microneedle show the needle in contact with the cell before application of pressure (0) and after needle movement of various distances (shown in microns across the top). The location of the needle shaft after movement of 125 μm and release from the cell by lifting is shown in the last frame of each panel. A cell treated with GST (top panel) is not deformed by pressure exerted by the needle. A cell treated with GST–C3 (bottom panel) is deformed by pressure more than the GST-treated cell; the needle tip moves further when a given amount of pressure is applied to the GST–C3 cell than when it is applied to the GST-treated cell. Bar, 125 μm. (B) Cell rigidity is plotted as force versus distance of deformation (see Materials and methods). Data points are averages and standard deviations (GST, black diamonds; GST–C3, gray squares). Linear regressions are plotted; slopes correspond to cell rigidity. *, significant difference from GST treatment (P < 0.0005). n > 25 for each treatment. The values for Y-27632 treatment are included in the text.
Figure 4.
Figure 4.
RhoA activity is elevated in mitosis. (A) RhoA activity was measured in lysates from control interphase adherent HeLa cells (Con), cells that had been rounded with trypsin (Tryp), or mitotic cells harvested by the knock-off method from untreated asynchronous cultures (Mit). RhoA immunoblots show RhoA protein levels in pull-downs (Active RhoA) and lysates (Total RhoA). *, significant difference from control (for all panels) (P < 0.05). Bars represent SEM. All data shown are representative of at least five replicate experiments. (B) HeLa cells were treated with nocodazole for 30 min (30'NZ) or overnight. For overnight treatment, rounded mitotic cells were harvested by knock-off (NZMit), with the remaining adherent cells in interphase (ON-NZ). (C) HeLa cells were treated with taxol for 30 min (30'TX) or overnight. Rounded mitotic cells (TXMit) that had accumulated during the overnight treatment were harvested by knock-off. Remaining interphase cells comprised the overnight interphase (ON-TX) sample. (D) HeLa cells were synchronized in G1 by a single thymidine block followed by a brief nocodazole block and then a washout to allow progression through mitosis and into G1 of interphase. Cells were synchronized in S phase with a double thymidine block, and in G2 with a double thymidine block followed by a 5-h washout. Rounded mitotic cells were harvested from cultures that were treated with taxol for 4 h after washout from a single thymidine block. (E) Staining of HeLa cell nuclei with Hoechst shows normal morphology with all treatments. (E, a) Untreated interphase cell, (b) untreated mitotic cell, (c–f) treated with 10 nM taxol, (c) 30' treatment (interphase cell), (d) overnight treatment (interphase cell), (e) overnight treatment (mitotic cell), (f) overnight treatment (cells harvested as rounded mitotic cells and fixed 7 h after washout, now interphase). Note that taxol does not cause the nuclear condensation characteristic of apoptosis at the concentration and time course used. Bar, 15 μm.
Figure 5.
Figure 5.
p190RhoGAP is tyrosine dephosphorylated, serine/threonine phosphorylated, and decreased in activity in mitosis. (A) p190RhoGAP was immunoprecipitated from HeLa cells and probed, stripped, and reprobed for p190RhoGAP, phosphotyrosine, and coassociation with p120RasGAP. Note the decrease in phosphotyrosine and p120RasGAP associated with mitotic p190RhoGAP, and the electrophoretic mobility shift in mitosis. (B) p190RhoGAP was immunoprecipitated from interphase and mitotic HeLa cells, and immunoprecipitates were washed into PP1 reaction buffer and divided into four equal samples. One sample was not treated (−); the remaining three each received 1 U PP1 phosphatase. The serine/threonine phosphatase inhibitor okadaic acid (PP1 + O) and the tyrosine phosphatase inhibitor vanadate (PP1 + V) were added to one sample each before PP1 was added. Note that the retarded electrophoretic mobility of mitotic p190RhoGAP is restored to the interphase mobility by PP1 treatment. (C) p190RhoGAP activity is lower in mitosis than in interphase as assayed for RhoA GAP activity. PP1 phosphatase treatment increases the activity of mitotic p190RhoGAP. Graphed are means from six experiments. Bars represent the SEM. *, significant difference from Interphase (−); **, significant difference from Mitosis (−) (P < 0.05).
Figure 6.
Figure 6.
Transient overexpression of GFP–p190RhoGAP does not block mitotic cell rounding. At 24 h after transfection, HeLa cells were fixed, stained, imaged, and measured as for Figs. 1 and 2. Also measured was the total GFP fluorescence within a circular region of fixed size, which comprised much of the cytoplasm. (A) A metaphase cell expressing GFP–p190RhoGAP has undergone mitotic cell rounding to a similar extent as a neighboring cell expressing little or no GFP–p190RhoGAP. (B) The extent of mitotic cell rounding, measured as cell diameter, perimeter, and area, was plotted against the intensity of the cytoplasmic GFP signal. GFP fluorescence is not predictive of the three cell rounding measurements.
Figure 7.
Figure 7.
RhoA and Rho-kinase are required for actin reorganization, cortical rigidity, and cortical retraction during mitotic cell rounding. Black arrows denote direct or indirect stimulation as shown here or in the literature. Gray arrows represent possible causal relationships. Black lines followed by bars denote inhibition.

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