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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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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.
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fig6: 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.

Mentions: The results presented above suggest that mitotic down-regulation of p190RhoGAP plays a role in RhoA-dependent mitotic cell rounding. To test whether this is true, we attempted to override mitotic down-regulation of p190RhoGAP. GFP-tagged wild-type p190RhoGAP was transiently overexpressed in HeLa cells and mitotic cell rounding was measured using images of cells in metaphase of mitosis. Imaging demonstrated that cells expressing GFP–p190RhoGAP undergo comparable mitotic cell rounding to nonexpressors (Fig. 6 A). Quantitation also revealed that the level of expression of p190RhoGAP was not predictive of cell size at the endpoint of mitotic cell rounding (Fig. 6 B). Therefore, using this method, we were unable to show that mitotic down-regulation of p190RhoGAP contributes to mitotic cell rounding.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: 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.
Mentions: The results presented above suggest that mitotic down-regulation of p190RhoGAP plays a role in RhoA-dependent mitotic cell rounding. To test whether this is true, we attempted to override mitotic down-regulation of p190RhoGAP. GFP-tagged wild-type p190RhoGAP was transiently overexpressed in HeLa cells and mitotic cell rounding was measured using images of cells in metaphase of mitosis. Imaging demonstrated that cells expressing GFP–p190RhoGAP undergo comparable mitotic cell rounding to nonexpressors (Fig. 6 A). Quantitation also revealed that the level of expression of p190RhoGAP was not predictive of cell size at the endpoint of mitotic cell rounding (Fig. 6 B). Therefore, using this method, we were unable to show that mitotic down-regulation of p190RhoGAP contributes to 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