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RhoA is required for cortical retraction and rigidity during mitotic cell rounding.

Maddox AS, Burridge K - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. akshaub@med.unc.edu

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.

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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.
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fig3: 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.

Mentions: In addition to cell rounding, entry into mitosis is also accompanied by an increase in cell rigidity (Mitchison and Swann, 1955; Yoneda and Dan, 1972; Matzke et al., 2001). Because it seemed likely that the same reorganization of the cortical cytoskeleton is required for cortical retraction during cell rounding and mitotic cortical rigidity, we hypothesized that RhoA is required for both processes. To determine whether RhoA is required for cells to become more rigid as they enter mitosis, we treated cells with C3, as for Fig. 1, and tested the rigidity of preanaphase mitotic cells (identified as having a metaphase plate of chromosomes by phase contrast imaging). To do this, a micromanipulation needle was brought into contact with the subject cell, and controlled increments of pressure were exerted on the cell by moving the needle shaft set distances. Images of the location of the needle tip and cell were captured and used to quantify the cells' resistance to pressure. Control mitotic cells resist pressure; they are not deformed by the needle (Fig. 3 A, top). Under the same amounts of pressure, a C3-treated mitotic cell is deformed visibly by the needle (Fig. 3 A, bottom). Cell rigidity is significantly decreased by the C3 treatment (Fig. 3 B; GST: −0.119, GST–C3: −0.210; see Materials and methods for quantitation details). Mitotic cells treated with Y-27632 to inhibit Rho-kinase are significantly less rigid than control cells (Control: −0.237 ± 0.098; Y-27632: −0.425 ± 0.14*; an asterisk indicates a significant difference from control; P < 0.0005.) From these results, we conclude that RhoA and Rho-kinase are required for both cortical retraction and increases in cortical rigidity as cells enter mitosis, and that these processes are likely linked.


RhoA is required for cortical retraction and rigidity during mitotic cell rounding.

Maddox AS, Burridge K - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: In addition to cell rounding, entry into mitosis is also accompanied by an increase in cell rigidity (Mitchison and Swann, 1955; Yoneda and Dan, 1972; Matzke et al., 2001). Because it seemed likely that the same reorganization of the cortical cytoskeleton is required for cortical retraction during cell rounding and mitotic cortical rigidity, we hypothesized that RhoA is required for both processes. To determine whether RhoA is required for cells to become more rigid as they enter mitosis, we treated cells with C3, as for Fig. 1, and tested the rigidity of preanaphase mitotic cells (identified as having a metaphase plate of chromosomes by phase contrast imaging). To do this, a micromanipulation needle was brought into contact with the subject cell, and controlled increments of pressure were exerted on the cell by moving the needle shaft set distances. Images of the location of the needle tip and cell were captured and used to quantify the cells' resistance to pressure. Control mitotic cells resist pressure; they are not deformed by the needle (Fig. 3 A, top). Under the same amounts of pressure, a C3-treated mitotic cell is deformed visibly by the needle (Fig. 3 A, bottom). Cell rigidity is significantly decreased by the C3 treatment (Fig. 3 B; GST: −0.119, GST–C3: −0.210; see Materials and methods for quantitation details). Mitotic cells treated with Y-27632 to inhibit Rho-kinase are significantly less rigid than control cells (Control: −0.237 ± 0.098; Y-27632: −0.425 ± 0.14*; an asterisk indicates a significant difference from control; P < 0.0005.) From these results, we conclude that RhoA and Rho-kinase are required for both cortical retraction and increases in cortical rigidity as cells enter mitosis, and that these processes are likely linked.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. akshaub@med.unc.edu

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.

Show MeSH
Related in: MedlinePlus