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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 activity is elevated in mitosis. (A) RhoA activity was measured in lysates from control interphase adherent HeLa cells (Con), cells that had been rounded with trypsin (Tryp), or mitotic cells harvested by the knock-off method from untreated asynchronous cultures (Mit). RhoA immunoblots show RhoA protein levels in pull-downs (Active RhoA) and lysates (Total RhoA). *, significant difference from control (for all panels) (P < 0.05). Bars represent SEM. All data shown are representative of at least five replicate experiments. (B) HeLa cells were treated with nocodazole for 30 min (30'NZ) or overnight. For overnight treatment, rounded mitotic cells were harvested by knock-off (NZMit), with the remaining adherent cells in interphase (ON-NZ). (C) HeLa cells were treated with taxol for 30 min (30'TX) or overnight. Rounded mitotic cells (TXMit) that had accumulated during the overnight treatment were harvested by knock-off. Remaining interphase cells comprised the overnight interphase (ON-TX) sample. (D) HeLa cells were synchronized in G1 by a single thymidine block followed by a brief nocodazole block and then a washout to allow progression through mitosis and into G1 of interphase. Cells were synchronized in S phase with a double thymidine block, and in G2 with a double thymidine block followed by a 5-h washout. Rounded mitotic cells were harvested from cultures that were treated with taxol for 4 h after washout from a single thymidine block. (E) Staining of HeLa cell nuclei with Hoechst shows normal morphology with all treatments. (E, a) Untreated interphase cell, (b) untreated mitotic cell, (c–f) treated with 10 nM taxol, (c) 30' treatment (interphase cell), (d) overnight treatment (interphase cell), (e) overnight treatment (mitotic cell), (f) overnight treatment (cells harvested as rounded mitotic cells and fixed 7 h after washout, now interphase). Note that taxol does not cause the nuclear condensation characteristic of apoptosis at the concentration and time course used. Bar, 15 μm.
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fig4: RhoA activity is elevated in mitosis. (A) RhoA activity was measured in lysates from control interphase adherent HeLa cells (Con), cells that had been rounded with trypsin (Tryp), or mitotic cells harvested by the knock-off method from untreated asynchronous cultures (Mit). RhoA immunoblots show RhoA protein levels in pull-downs (Active RhoA) and lysates (Total RhoA). *, significant difference from control (for all panels) (P < 0.05). Bars represent SEM. All data shown are representative of at least five replicate experiments. (B) HeLa cells were treated with nocodazole for 30 min (30'NZ) or overnight. For overnight treatment, rounded mitotic cells were harvested by knock-off (NZMit), with the remaining adherent cells in interphase (ON-NZ). (C) HeLa cells were treated with taxol for 30 min (30'TX) or overnight. Rounded mitotic cells (TXMit) that had accumulated during the overnight treatment were harvested by knock-off. Remaining interphase cells comprised the overnight interphase (ON-TX) sample. (D) HeLa cells were synchronized in G1 by a single thymidine block followed by a brief nocodazole block and then a washout to allow progression through mitosis and into G1 of interphase. Cells were synchronized in S phase with a double thymidine block, and in G2 with a double thymidine block followed by a 5-h washout. Rounded mitotic cells were harvested from cultures that were treated with taxol for 4 h after washout from a single thymidine block. (E) Staining of HeLa cell nuclei with Hoechst shows normal morphology with all treatments. (E, a) Untreated interphase cell, (b) untreated mitotic cell, (c–f) treated with 10 nM taxol, (c) 30' treatment (interphase cell), (d) overnight treatment (interphase cell), (e) overnight treatment (mitotic cell), (f) overnight treatment (cells harvested as rounded mitotic cells and fixed 7 h after washout, now interphase). Note that taxol does not cause the nuclear condensation characteristic of apoptosis at the concentration and time course used. Bar, 15 μm.

Mentions: The fact that RhoA is required for both cortical retraction and increases in cortical rigidity as cells enter mitosis suggests that RhoA activity increases during this cell cycle transition. To test this, we compared the activity of RhoA in spread interphase cells with that in rounded mitotic cells. Rounded mitotic HeLa cells were harvested from untreated asynchronous cultures by mechanical disruption. The adherent cells remaining after this “knock-off” procedure were in interphase and comprised the control sample (Fig. 4 E, a and b). To determine the degree of RhoA activation in each condition, we isolated active, GTP-bound RhoA from cell lysates using an affinity matrix consisting of the RhoA-binding domain of the RhoA effector Rhotekin fused to GST and immobilized on sepharose beads (Ren et al., 1999). Active RhoA associated with the beads, and total RhoA in lysates was detected by immunoblotting. We found that, whereas the total level of RhoA did not change, there was significantly more active RhoA in lysates from mitotic cells than in lysates prepared from interphase controls (Fig. 4 A). The amount of active RhoA was also elevated in trypsin-rounded interphase cells, as has been seen (Ren et al., 1999).


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

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

RhoA activity is elevated in mitosis. (A) RhoA activity was measured in lysates from control interphase adherent HeLa cells (Con), cells that had been rounded with trypsin (Tryp), or mitotic cells harvested by the knock-off method from untreated asynchronous cultures (Mit). RhoA immunoblots show RhoA protein levels in pull-downs (Active RhoA) and lysates (Total RhoA). *, significant difference from control (for all panels) (P < 0.05). Bars represent SEM. All data shown are representative of at least five replicate experiments. (B) HeLa cells were treated with nocodazole for 30 min (30'NZ) or overnight. For overnight treatment, rounded mitotic cells were harvested by knock-off (NZMit), with the remaining adherent cells in interphase (ON-NZ). (C) HeLa cells were treated with taxol for 30 min (30'TX) or overnight. Rounded mitotic cells (TXMit) that had accumulated during the overnight treatment were harvested by knock-off. Remaining interphase cells comprised the overnight interphase (ON-TX) sample. (D) HeLa cells were synchronized in G1 by a single thymidine block followed by a brief nocodazole block and then a washout to allow progression through mitosis and into G1 of interphase. Cells were synchronized in S phase with a double thymidine block, and in G2 with a double thymidine block followed by a 5-h washout. Rounded mitotic cells were harvested from cultures that were treated with taxol for 4 h after washout from a single thymidine block. (E) Staining of HeLa cell nuclei with Hoechst shows normal morphology with all treatments. (E, a) Untreated interphase cell, (b) untreated mitotic cell, (c–f) treated with 10 nM taxol, (c) 30' treatment (interphase cell), (d) overnight treatment (interphase cell), (e) overnight treatment (mitotic cell), (f) overnight treatment (cells harvested as rounded mitotic cells and fixed 7 h after washout, now interphase). Note that taxol does not cause the nuclear condensation characteristic of apoptosis at the concentration and time course used. Bar, 15 μm.
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Related In: Results  -  Collection

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fig4: RhoA activity is elevated in mitosis. (A) RhoA activity was measured in lysates from control interphase adherent HeLa cells (Con), cells that had been rounded with trypsin (Tryp), or mitotic cells harvested by the knock-off method from untreated asynchronous cultures (Mit). RhoA immunoblots show RhoA protein levels in pull-downs (Active RhoA) and lysates (Total RhoA). *, significant difference from control (for all panels) (P < 0.05). Bars represent SEM. All data shown are representative of at least five replicate experiments. (B) HeLa cells were treated with nocodazole for 30 min (30'NZ) or overnight. For overnight treatment, rounded mitotic cells were harvested by knock-off (NZMit), with the remaining adherent cells in interphase (ON-NZ). (C) HeLa cells were treated with taxol for 30 min (30'TX) or overnight. Rounded mitotic cells (TXMit) that had accumulated during the overnight treatment were harvested by knock-off. Remaining interphase cells comprised the overnight interphase (ON-TX) sample. (D) HeLa cells were synchronized in G1 by a single thymidine block followed by a brief nocodazole block and then a washout to allow progression through mitosis and into G1 of interphase. Cells were synchronized in S phase with a double thymidine block, and in G2 with a double thymidine block followed by a 5-h washout. Rounded mitotic cells were harvested from cultures that were treated with taxol for 4 h after washout from a single thymidine block. (E) Staining of HeLa cell nuclei with Hoechst shows normal morphology with all treatments. (E, a) Untreated interphase cell, (b) untreated mitotic cell, (c–f) treated with 10 nM taxol, (c) 30' treatment (interphase cell), (d) overnight treatment (interphase cell), (e) overnight treatment (mitotic cell), (f) overnight treatment (cells harvested as rounded mitotic cells and fixed 7 h after washout, now interphase). Note that taxol does not cause the nuclear condensation characteristic of apoptosis at the concentration and time course used. Bar, 15 μm.
Mentions: The fact that RhoA is required for both cortical retraction and increases in cortical rigidity as cells enter mitosis suggests that RhoA activity increases during this cell cycle transition. To test this, we compared the activity of RhoA in spread interphase cells with that in rounded mitotic cells. Rounded mitotic HeLa cells were harvested from untreated asynchronous cultures by mechanical disruption. The adherent cells remaining after this “knock-off” procedure were in interphase and comprised the control sample (Fig. 4 E, a and b). To determine the degree of RhoA activation in each condition, we isolated active, GTP-bound RhoA from cell lysates using an affinity matrix consisting of the RhoA-binding domain of the RhoA effector Rhotekin fused to GST and immobilized on sepharose beads (Ren et al., 1999). Active RhoA associated with the beads, and total RhoA in lysates was detected by immunoblotting. We found that, whereas the total level of RhoA did not change, there was significantly more active RhoA in lysates from mitotic cells than in lysates prepared from interphase controls (Fig. 4 A). The amount of active RhoA was also elevated in trypsin-rounded interphase cells, as has been seen (Ren et al., 1999).

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