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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 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).
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fig1: 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).

Mentions: Mitotic cell rounding is comprised of at least two independent processes: de-adhesion and cortical retraction. De-adhesion is the dissolution of stress fibers and focal adhesions and the dispersal of the component proteins. This has been described for several stress fiber and focal adhesion proteins (Sanger et al., 1987; Hock et al., 1989; Sanger et al., 1989). During cortical retraction, the cell cortex changes from well spread and flattened to spherical and refractile by phase contrast imaging. To examine whether RhoA has a role in either of these processes, we treated HeLa cells with a GST fusion with C3 toxin (C3), an inhibitor of RhoA. This treatment significantly reduces RhoA-GTP levels but does not inhibit the related GTPase Rac1 (unpublished data). To test for a role in de-adhesion, we first stained control and C3-treated cells with an antibody to paxillin, a component of focal adhesions, and with phalloidin to follow changes in the actin cytoskeleton. To ensure that cells were at or near the endpoint of mitotic rounding, only cells with a metaphase plate of condensed, aligned chromosomes were imaged (PFig. 1, O and P). We found that in C3-treated cells and GST-treated control cells, paxillin is diffusely localized in mitotic cells (Fig. 1, compare I with K and J with L). Also, actin stress fibers are absent in mitotic C3-treated cells, as in control cells (Fig. 1, compare E with G and F with H). This suggests that RhoA activity is not required for de-adhesion.


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

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

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).
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Related In: Results  -  Collection

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fig1: 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).
Mentions: Mitotic cell rounding is comprised of at least two independent processes: de-adhesion and cortical retraction. De-adhesion is the dissolution of stress fibers and focal adhesions and the dispersal of the component proteins. This has been described for several stress fiber and focal adhesion proteins (Sanger et al., 1987; Hock et al., 1989; Sanger et al., 1989). During cortical retraction, the cell cortex changes from well spread and flattened to spherical and refractile by phase contrast imaging. To examine whether RhoA has a role in either of these processes, we treated HeLa cells with a GST fusion with C3 toxin (C3), an inhibitor of RhoA. This treatment significantly reduces RhoA-GTP levels but does not inhibit the related GTPase Rac1 (unpublished data). To test for a role in de-adhesion, we first stained control and C3-treated cells with an antibody to paxillin, a component of focal adhesions, and with phalloidin to follow changes in the actin cytoskeleton. To ensure that cells were at or near the endpoint of mitotic rounding, only cells with a metaphase plate of condensed, aligned chromosomes were imaged (PFig. 1, O and P). We found that in C3-treated cells and GST-treated control cells, paxillin is diffusely localized in mitotic cells (Fig. 1, compare I with K and J with L). Also, actin stress fibers are absent in mitotic C3-treated cells, as in control cells (Fig. 1, compare E with G and F with H). This suggests that RhoA activity is not required for de-adhesion.

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