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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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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).
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fig5: 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).

Mentions: Tyrosine phosphorylation of p190RhoGAP has been correlated with its activity (Fincham et al., 1999; Arthur et al., 2000; Dumenil et al., 2000). Immunoprecipitated p190RhoGAP bears less tyrosine phosphorylation in mitosis than in interphase (Fig. 5 A). Also, decreased p120RasGAP is present in p190RhoGAP immunoprecipitates in mitosis (Fig. 5 A). These findings suggest that mitotic p190RhoGAP has decreased activity (Trouliaris et al., 1995). Careful inspection of immunoprecipitated p190RhoGAP revealed that it migrates more slowly and as a more diffuse band in mitosis (Fig. 5, A and B). Changes in gel migration often reflect altered levels of serine/threonine phosphorylation. Therefore, immunoprecipitates were treated with protein phosphatase 1 (PP1). This treatment restored the mobility of mitotic p190RhoGAP to that of p190RhoGAP from interphase cells (Fig. 5 B). These results suggest that in mitosis, p190RhoGAP has decreased phosphotyrosine, decreased association with p120RasGAP, and an altered electrophoretic mobility due to increased phosphoserine or phosphothreonine.


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

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

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).
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fig5: 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).
Mentions: Tyrosine phosphorylation of p190RhoGAP has been correlated with its activity (Fincham et al., 1999; Arthur et al., 2000; Dumenil et al., 2000). Immunoprecipitated p190RhoGAP bears less tyrosine phosphorylation in mitosis than in interphase (Fig. 5 A). Also, decreased p120RasGAP is present in p190RhoGAP immunoprecipitates in mitosis (Fig. 5 A). These findings suggest that mitotic p190RhoGAP has decreased activity (Trouliaris et al., 1995). Careful inspection of immunoprecipitated p190RhoGAP revealed that it migrates more slowly and as a more diffuse band in mitosis (Fig. 5, A and B). Changes in gel migration often reflect altered levels of serine/threonine phosphorylation. Therefore, immunoprecipitates were treated with protein phosphatase 1 (PP1). This treatment restored the mobility of mitotic p190RhoGAP to that of p190RhoGAP from interphase cells (Fig. 5 B). These results suggest that in mitosis, p190RhoGAP has decreased phosphotyrosine, decreased association with p120RasGAP, and an altered electrophoretic mobility due to increased phosphoserine or phosphothreonine.

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