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Address correspondence to Amy Shaub Maddox, 12-026 Lineberger, UNC-CH Chapel Hill, NC 27599. Tel.: (919) 966-5783. Fax: (919) 966-1856. E-mail: akshaub{at}med.unc.edu
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Abstract |
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Key Words: mitotic cell rounding; mitosis; cortical rigidity; actin; RhoA
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Introduction |
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Mitotic cell rounding is accompanied by changes in the actin cytoskeleton. In interphase of many types of cultured cells, actin is predominantly organized into stress fibers that span the cytoplasm. Upon entry into mitosis, stress fibers disassemble and actin localizes primarily to the increasingly round cortex. Cramer and Mitchison (1997) showed that filamentous actin (F-actin) is required for coordinated retraction of the cell margin at the onset of mitosis, demonstrating that the actin cytoskeleton plays an active role in mitotic cell rounding. The enrichment of F-actin in the spherical cortex in mitosis could be favored by the cross-linking of actin filaments into a meshwork. Several actin-binding proteins can support such cross-linking, including filamin, spectrin, and -actinin. Evidence that actin cross-linking promotes a rounded morphology comes from Cortese et al. (1989). The inclusion of filamin in actin-containing vesicles caused the vesicles to become smooth and spherical upon actin polymerization, whereas an irregular, angular morphology occurred in the absence of filamin (Cortese et al., 1989). Adhesions to the substrate are also altered in mitosis but remain connected to the cell via retraction fibers, which are exposed as the cell rounds. Structural and signaling proteins resident to focal adhesions become diffusely localized within the cytoplasm (Sanger et al., 1987; Hock et al., 1989; Yamakita et al., 1999). Plating cells on flexible substrates revealed that intracellular tension transmitted to the substrate through focal adhesions decreases during entry into mitosis (Burton and Taylor, 1997). Here, we will refer to this disassembly of focal adhesions as de-adhesion.
The Rho family of small GTPases regulates actin organization and therefore cell shape (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998). One of the best-characterized members of this family is RhoA. Many RhoA effectors lead to remodeling of the actin cytoskeleton. The RhoA effector Rho-kinase stimulates the myosin II regulatory light chain (MLC)* directly by phosphorylation and indirectly by inhibition of myosin phosphatase (Amano et al., 1996; Kimura et al., 1996). Another RhoA effector, citron kinase, also activates MLC by phosphorylation (Matsumura et al., 2001). Activation of MLC leads to actomyosin contractility, bundling, and cross-linking of actin filaments, and thus the formation and maintenance of actin stress fibers (Chrzanowska-Wodnicka and Burridge, 1996). The RhoA effector mDia, which promotes actin filament bundling, also contributes to proper stress fiber formation (Watanabe et al., 1997, 1999). Additionally, RhoA activity regulates the actin cytoskeleton by affecting actin filament assembly dynamics. RhoA, via Rho-kinase, stimulates LIM-kinase (LIMK), which down-regulates the actin-severing protein cofilin by phosphorylation (Maekawa et al., 1999; Sumi et al., 1999).
Inhibition of RhoA by treatment with C3 toxin causes dissolution of stress fibers and cell rounding in interphase cells (Paterson et al., 1990; Wiegers et al., 1991). The latter is thought to occur because inhibition of RhoA results in decreased focal adhesions and substrate adhesions in general. When RhoA is inhibited with C3 in mitotic cells, the actomyosin cytokinetic furrow is blocked (Kishi et al., 1993). Likewise, Y-27632, a specific inhibitor of Rho-kinase, causes dissolution of stress fibers and retraction of the cell margin (Uehata et al., 1997), and blocks MLC phosphorylation and furrow ingression during cytokinesis (Kosako et al., 2000). Interestingly, in earlier stages of mitosis, C3 treatment resulted in the spreading of the treated prophase cell as it was pulled by neighboring cells in a confluent monolayer of epithelial cells (O'Connell et al., 1999). The authors suggest that RhoA regulates the "mechanical integrity and strength of the cortex" (O'Connell et al., 1999). We hypothesized that RhoA mediates mitotic reorganization of the actin cytoskeleton, and that this rearrangement promotes cortical rigidity in mitosis and mitotic cell rounding.
Here we examine the role of RhoA in mitotic cell rounding. We show that RhoA is required for cortical retraction, but not de-adhesion during rounding. RhoA is also required for increases in cortical rigidity as cells enter mitosis, suggesting that cortical retraction and increased cortical rigidity are linked processes. Consistent with a role during this cell cycle transition, RhoA activity is elevated in preanaphase mitotic cells compared with interphase cells. The negative regulator of RhoA, p190RhoGAP, is less active at metaphase than in interphase and this down-regulation may play a role in mitotic stimulation of RhoA.
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Results |
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Inhibition of Rho-kinase also results in defects in cortical retraction during mitotic cell rounding
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-27632treated 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-27632treated 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.
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Hoechst staining of DNA shows that cells treated with this low level of taxol (10 nM) were not undergoing apoptosis (Fig. 4 E, cf). Interphase cells treated with taxol for 812 h have normal nuclear morphology (Fig. 4 E, d). Mitotic taxol-treated cells had condensed chromatin before taxol washout (Fig. 4 E, e). At 7 h after taxol washout, nuclei have interphase morphology but are lobulated (Fig. 4 E, f). This may be due to the abnormal mitoses (multipolar spindles) that occur in taxol-treated cells (Brown et al., 1985).
We next examined whether RhoA activity is elevated at any other stage of the cell cycle besides the elevation that occurs during mitosis. We assayed RhoA activity in HeLa cells synchronized in G1, S phase, and G2 and compared RhoA activity at these times with that in mitosis and in asynchronous cells. There is no significant difference in RhoA activity among the various cell cycle phases other than in mitosis (Fig. 4 D). From these results, we conclude that RhoA activity increases as cells enter mitosis. Combined with our previous data, these results further suggest that increased RhoA activity leads to increased cortical rigidity and cortical retraction, which cause cell rounding at the onset of mitosis.
p190RhoGAP activity is lower in mitosis than in interphase
Rho proteins are regulated by two major families of proteins: guanine nucleotide exchange factors (GEFs), which activate Rho proteins by loading them with GTP, and GTPase activating proteins (GAPs), which inactivate Rho proteins by promoting their intrinsic GTP hydrolysis activity (for review see Bishop and Hall, 2000). Also, guanine nucleotide disassociation inhibitors interact with and inhibit Rho GTPases (Bishop and Hall, 2000). The mitotic increase in RhoA activity is most likely due either to the activation of a GEF or the inactivation of a GAP (Vincent and Settleman, 1999). The recent observation that the decrease in RhoA activity after integrin engagement is mediated by p190RhoGAP (Arthur et al., 2000) led us to investigate whether this negative regulatory protein was also responsible for changes in RhoA activity in mitosis, another incidence of adhesion dynamics.
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.
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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 GFPp190RhoGAP 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.
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Discussion |
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The results presented in Fig. 5 suggested that this regulation of p190RhoGAP plays a role in RhoA-dependent mitotic cell rounding. However, transient expression of p190RhoGAP did not block rounding (Fig. 6). Importantly, the mitotic cells that were measured likely expressed only low levels of p190RhoGAP and consequently may still have had adequate levels of active RhoA. Cells expressing high levels of GFPp190RhoGAP were highly retracted in interphase or were apoptotic, and it is doubtful that many of these cells entered mitosis. Also, it is possible that the exogenous p190RhoGAP was properly down-regulated, and that RhoA was activated sufficiently to elicit mitotic rounding. Therefore, this approach cannot conclusively address whether p190RhoGAP down-regulation in mitosis is required for mitotic cell rounding. We also found that mitotic cell rounding was not blocked in Rat1 cells stably overexpressing p190RhoGAP at three to five times the endogenous levels (unpublished data). However, treatment with C3 revealed that mitotic cell rounding is RhoA independent in these cells. This indicates that, at least in Rat1 cells, there is an additional Rho-independent mechanism for this process. Interestingly, both the elevation of p190RhoGAP and C3 treatment increased blebbing in mitotic Rat1 cells, consistent with a role for RhoA in regulating cortical rigidity during mitosis in these cells. Therefore, we conclude that mitotic activation of RhoA may be due in part to p190RhoGAP down-regulation, but that a Rho GEF likely contributes to mitotic RhoA activation and cell rounding (Fig. 7).
How does RhoA activation promote mitotic cell rounding?
Studies demonstrating mitotic cortical rigidity (Mitchison and Swann, 1955; Yoneda and Dan, 1972; Matzke et al., 2001) have not addressed its cause. Among the factors that could affect this physical characteristic are cytoskeletal contractility under the cortex, actin filament cross-linking, and the degree of coupling between the cortical cytoskeleton and the membrane. RhoA, a major regulator of the actin cytoskeleton, can affect all three of these characteristics. We propose that RhoA and Rho-kinase promote mitotic cortical retraction and rigidity through regulation of the actin cytoskeleton (Fig. 7). RhoA regulates the actin cytoskeleton in many ways. Most attention has been directed toward the RhoA-dependent Rho-kinase stimulation of phosphorylation of MLC and the consequent stimulation of myosin contractility (Amano et al., 1996; Kimura et al., 1996). It was recently shown that the rounded shape of FAK-/- cells is alleviated by inhibition of Rho-kinase by Y-27632, another example where a rounded morphology is dependent on RhoA and Rho-kinase activity (Chen et al., 2002). Yamakita et al. (1994) showed that in preanaphase mitotic cells, the majority of MLC is phosphorylated (possibly by Cdc2 [Satterwhite et al., 1992] or a conventional PKC PKC [Varlamova et al., 2001]) on residues that confer inhibition. Inhibitory phosphorylation of MLC blocks phosphorylation on stimulatory residues (Nishikawa et al., 1984). Therefore, even in the presence of high RhoA activity in mitosis, inhibitory phosphorylation would block stimulatory phosphorylation by Rho-kinase. Interestingly, Yamakita et al. (1994) report that although 31% of MLC is phosphorylated early in mitosis, only 15% of MLC is phosphorylated on inhibitory residues at this time. This would seem to allow for the possibility that some MLC in mitosis bears stimulatory phosphorylation and is actively contractile. In addition, Shuster and Burgess (1999) showed that at no time does cortical MLC bear inhibitory phosphorylation, and that in metaphase of mitosis, some cortical MLC bears stimulatory phosphorylation. Further evidence for the presence of myosin contractility in preanaphase mitosis comes from Sanger et al. (1989), who performed time-lapse microscopy of mitosis in cells that had been microinjected with fluorescently labeled MLC. The labeled myosin incorporated into and localized to puncta along stress fibers. As cells entered mitosis and underwent mitotic cell rounding, stress fibers shortened and individual myosin-containing puncta along stress fibers became closer together. Microinjected fluorescent -actinin had a similar behavior (Sanger et al., 1987). This observation of actomyosin contractility at this stage of the cell cycle demonstrates that myosin contractility could contribute to the state of the cortical actin cytoskeleton at this time.
Several other Rho-kinase substrates also regulate the actin cytoskeleton. Adducin, which links F-actin to the plasma membrane through spectrin, is stimulated by phosphorylation by Rho-kinase (Kimura et al., 1998; Fukata et al., 1999). The resulting enhancement of coupling between the actin cytoskeleton and the spectrin-based membrane cytoskeleton could promote mitotic cortical rigidity and rounding. Cofilin, an actin-severing protein that promotes depolymerization of F-actin, is down-regulated by RhoA signaling through Rho-kinase and inhibitory phosphorylation by LIMK (Maekawa et al., 1999). It has recently been shown that cofilin activity in mitosis is lower than in interphase, but then decreases further before increasing to interphase levels as cells undergo cytokinesis (Amano et al., 2002). Part of this multiphasic regulation has been shown to be independent of Rho-kinase and LIMK (Amano et al., 2002). However, the possibility exists that elevated RhoA activity contributes to the down-regulation of cofilin in mitosis. This would lead to a stabilization of the actin cytoskeleton and rigidity in rounded mitotic cells.
The formation and maintenance of stress fibers and focal adhesions are stimulated by and require RhoA activity (Chardin et al., 1989; Ridley and Hall, 1992). However, in mitosis, stress fibers and the associated focal adhesions disassemble in the presence of high RhoA activity. Therefore, the disassembly of these structures must be driven by a mitotic pathway independent of RhoA activity. One candidate mechanism is down-regulation of myosin contractility, which promotes these structures. However, as discussed above, the state of myosin activity during mitotic cell rounding has not been clearly defined. Another possibility is that an actin-binding protein disrupts the interaction of actin with myosin. Perhaps mitotic regulation of caldesmon causes stress fiber disassembly in the presence of high RhoA activity (Yamashiro et al., 1991). Because actomyosin contractility is required to maintain focal adhesions, stress fiber disassembly alone may be sufficient for focal adhesion disassembly in mitosis. However, mitotic phosphorylation of the cytoplasmic focal adhesion proteins FAK, p130cas, and paxillin could contribute to focal adhesion disassembly (Yamaguchi et al., 1997; Yamakita et al., 1999). Alternatively, it is possible that there are spatially distinct pools of RhoA activity in the cell during mitotic cell rounding. We are currently investigating the mechanism for the mitotic disassembly of stress fibers and focal adhesions in the presence of high overall RhoA activity.
It has long been known that most animal cells round up during mitosis. Why is mitotic cell rounding such a universal phenomenon? Although there is no evidence for a "cell rounding checkpoint," Rieder et al. (1994) observed that the height of PtK1 epithelial cells (i.e., their degree of rounding) correlated inversely with the time taken to progress from nuclear envelope breakdown to anaphase onset. This suggests that cell rounding facilitates the assembly and function of the mitotic spindle. Mitotic cell rounding might prevent aneuploidy and delays in mitosis by helping to center the spindle, bringing the spindle poles together, and preventing chromosome mono-orientation. We propose that in mitosis, RhoA, via Rho-kinase, causes the cortex to become rigid and spherical in shape, and thereby promotes the fidelity and speed of chromosome alignment and segregation.
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Materials and methods |
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For C3 treatment, 0.28 µg GSTC3 (prepared as in Worthylake et al., 2001) per 24-well plate well was incubated in serum-free medium and 1.7 µl LipofectAmine (Invitrogen) and then added to cells in serum-free medium. Cells were incubated with GSTC3 or GST alone for 90 min, fixed, and processed for microscopy. For Y-27632 treatment, cells were treated with 10 µM Y-27632 (Welfide Corp.) for 1 h (2 h for rigidity experiments) in growth medium.
Determination of relative cell rigidity
HeLa cells were treated with GST or GSTC3 as for Fig. 1, or with Y-27632 as above. Cells in metaphase of mitosis were identified by phase contrast microscopy (Nikon or Carl Zeiss MicroImaging, Inc.) as having a metaphase plate of condensed chromatin. A glass microneedle was brought laterally to the surface of the cell. Using the micromanipulator (Narishige or Eppendorf) manual control dials, the microneedle shaft was moved laterally to standardized distances (0, 25, 50, 75, 100, and 125 µm), and images of the cell and the needle tip were captured for each distance of shaft movement, which corresponds to the amount of pressure applied. The location of the microneedle tip against the cell was plotted. Stiffness, or rigidity, is defined as force/change in length. Cell rigidity is plotted as the distance of deformation per amount of force, or pressure, applied. The same needle was used for control and untreated cell measurements within each experiment.
Immunofluorescence and immunoblotting
To visualize actin and DNA, cells plated on glass coverslips were fixed for 10 min in 3.7% formaldehyde in PBS, permeabilized for 5 min in 0.5% Triton X-100 in TBS (150 mM NaCl, 50 mM Tris-Cl, pH 7.6), and washed in TBS containing 0.1% azide. Coverslips were washed in TBS + azide. Cells were labeled with Alexa®594 phalloidin (Molecular Probes), Hoechst 33342 (Molecular Probes), or a monoclonal anti-paxillin antibody (BD Biosciences) and an AlexaFluor®488 secondary antimouse antibody (Molecular Probes). Images were collected using a Carl Zeiss MicroImaging, Inc. Axiophot with a Micromax cooled CCD camera (Princeton Instruments) driven by Metamorph software (Universal Imaging Corp.). We used primary antibodies to RhoA, p190RhoGAP, p120RasGAP, and HRP-conjugated anti-phosphotyrosine (mouse monoclonals; BD Biosciences) and an HRP-conjugated antimouse secondary antibody (Jackson ImmunoResearch Laboratories).
RhoA activity assay
The RhoA activity pulldown assay was performed as published (Arthur and Burridge, 2001).
Immunoprecipitations
HeLa cells were treated overnight with 10 nM taxol, and rounded mitotic cells were collected by knock-off (mechanical disruption). The remaining adherent cells comprised the interphase sample. For immunoblotting, coimmunoprecipitation with p120RasGAP, and PP1 treatment, cells were lysed in a modified RIPA buffer (150 mM NaCl, 6 mM Na2HPO4, 4 mM NaH2PO4, 1% deoxycholic acid, 1% NP-40, 0.1% SDS, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM orthovanadate, 1 mM PMSF, 50 nM calyculin A) and precleared with antimouse-conjugated beads (Sigma-Aldrich). Lysates were clarified by centrifugation, protein concentrations were determined and adjusted to equal levels, and lysates were incubated with anti-p190RhoGAP (3 µl/500 µg total protein; BD Biosciences) at 4°C for 1.5 h. Antimouse IgG beads were added and incubated for an additional 30 min. Beads were washed three times in lysis buffer and prepared for SDS-PAGE by boiling in sample buffer. For GAP assays, immunoprecipitations were performed as published (Fincham et al., 1999).
PP1 treatment
Sedimented p190RhoGAP on beads from immunoprecipitations, as described above, was washed two times into reaction buffer (50 mM imidazole, pH 7.0, 0.25 mM MnCl2, 5 mM DTT, 100 mM KCl, 2 mM MgCl2, 0.2 mg/ml BSA) and divided equally for the following treatments. One sample was left untreated, one sample received PP1 (Calbiochem) alone (1 U), one sample received okadaic acid (final concentration 4 µM) before addition of PP1, and the fourth sample received vanadate (final concentration 2 mM) before PP1. Reactions were incubated at 30°C for 30 min and stopped by the addition of sample buffer and boiling.
GAP activity assay
GAP assays were performed as published (Fincham et al., 1999).
Transfections
Transient transfections were performed with LipofectAmine Plus (GIBCO BRL) according to the manufacturer's instructions.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grant GM29860.
Submitted: 24 July 2002
Revised: 11 December 2002
Accepted: 12 December 2002
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References |
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