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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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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).
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fig2: 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).

Mentions: Rho-kinase is a RhoA effector that plays an important role in transducing signals from RhoA to the actin cytoskeleton (Leung et al., 1995; Ishizaki et al., 1996; Kimura et al., 1996). Therefore, we investigated whether Rho-kinase is also required for cortical retraction during mitotic cell rounding. HeLa cells were treated with the Rho-kinase inhibitor Y-27632, and cells in metaphase of mitosis were assessed for degree of rounding by measuring diameter, perimeter, and area. As was the case for the C3-treated cells, by all three criteria, cortical retraction was significantly inhibited in Y-27632–treated cells compared with controls (Fig. 2 G; Diameter: Control, 17.0 ± 2.2 μm; Y-27632, 19.9 ± 3.2 μm; Perimeter: Control, 53.7 ± 6.8 μm; Y-27632, 61.9 ± 10.6 μm; Area: Control, 206.6 ± 42.2 μm2; Y-27632, 254.8 ± 60.7 μm2). Phalloidin staining of F-actin reveals that the intense circular band on actin in control mitotic cells is absent in Y-27632–treated cells (Fig. 2, C and D). These results show that Rho-kinase is likely a key effector that mediates the RhoA dependency of cortical retraction during mitotic cell rounding.


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

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

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172639&req=5

fig2: 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).
Mentions: Rho-kinase is a RhoA effector that plays an important role in transducing signals from RhoA to the actin cytoskeleton (Leung et al., 1995; Ishizaki et al., 1996; Kimura et al., 1996). Therefore, we investigated whether Rho-kinase is also required for cortical retraction during mitotic cell rounding. HeLa cells were treated with the Rho-kinase inhibitor Y-27632, and cells in metaphase of mitosis were assessed for degree of rounding by measuring diameter, perimeter, and area. As was the case for the C3-treated cells, by all three criteria, cortical retraction was significantly inhibited in Y-27632–treated cells compared with controls (Fig. 2 G; Diameter: Control, 17.0 ± 2.2 μm; Y-27632, 19.9 ± 3.2 μm; Perimeter: Control, 53.7 ± 6.8 μm; Y-27632, 61.9 ± 10.6 μm; Area: Control, 206.6 ± 42.2 μm2; Y-27632, 254.8 ± 60.7 μm2). Phalloidin staining of F-actin reveals that the intense circular band on actin in control mitotic cells is absent in Y-27632–treated cells (Fig. 2, C and D). These results show that Rho-kinase is likely a key effector that mediates the RhoA dependency of cortical retraction during mitotic cell rounding.

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