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1 Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan
2 Department of Dermatology, Mie University, Faculty of Medicine, Tsu, Mie 514-8507, Japan
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Introduction |
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The small GTP-binding protein Rho controls organization of actin cytoskeleton (Narumiya et al. 1997; Kaibuchi et al. 1999). Inhibition of endogenous Rho by botulinum ADP-ribosyltransferase C3 blocked cytokinesis in Xenopus embryo (Kishi et al. 1993) and sand dollar eggs (Mabuchi et al. 1993). Moreover, C3 treatment induced ectopic cleavage furrow in normal rat kidney cells and perturb cytokinesis in HeLa cells (O’Connell et al. 1999; Eda et al. 2001). Rho-kinase/ROCK, one of the downstream effectors of Rho, regulates not only the formation of stress fibers and focal adhesion complexes (Leung et al. 1996; Amano et al. 1997; Ishizaki et al. 1997) but also the contractility by inactivating the myosin II light chain phosphatase (MLCP) which inhibit the myosin II activity (Kimura et al. 1996; Kawano et al. 1999). Expression of the dominant-negative form of Rho-kinase/ROCK2 inhibits the cytokinesis of Xenopus embryo and mammalian cells (Yasui et al. 1998). Therefore Rho-kinase/ROCK pathway is required for the reorganization of cleavage activities during cell division.
Aurora-B (also called AIM-1) is an evolutionally conserved protein kinase localizing on centromeres from prophase to metaphase-anaphase transition, thereby relocalizes to the spindle midzone and midbody from anaphase to cytokinesis (Adams et al. 2001; Nigg 2001; Carmena & Earnshaw 2003). A kinase-inactive form of Aurora-B inhibits the formation of cleavage furrow without affecting nuclear division (Tatsuka et al. 1998; Terada et al. 1998). AIR-2, a Caenorhabditis elegans homologue of mammalian Aurora-B, is required for cytokinesis through the appropriate localization of ZEN-4 which encodes a homologue of the mammalian mitotic kinesin-like protein-1 (MKLP-1; Severson et al. 2000). By using RNA interference (RNAi) of Aurora-B or Aurora kinase inhibitors, it has been demonstrated that human Aurora-B is required not only for mitotic phosphorylation of histone H3, chromosome alignment, and chromosome segregation, but also cytokinesis (Ditchfield et al. 2003; Hauf et al. 2003), consistent with earlier observations in C. elegans embryos and Drosophila melanogaster (Adams et al. 2001).
To date, the putative substrates for Rho-kinase/ROCK are myosin light chain (MLC; Amano et al. 1996), myosin binding subunit of myosin phosphatase (MBS; Kimura et al. 1996), ezrin/radixin/moesin (ERM) proteins (Matsui et al. 1998) and type III intermediate filaments including vimentin, glial fibrillary acidic protein (GFAP), and desmin (Kosako et al. 1997, 1999; Yasui et al. 1998; Goto et al. 1998; Kawajiri et al. 2003). In contrast, the putative substrates for Aurora-B are histone H3 (Hsu et al. 2000; Giet & Glover 2001; Murnion et al. 2001; Goto et al. 2002), centromere protein A (CENP-A; Zeitlin et al. 2001), inner centromere protein (INCENP; Bishop & Schumacher 2002), MLC (Murata-Hori et al. 2000), topoisomerase II (Morrison et al. 2002), MgcRacGAP (Minoshima et al. 2003; Ban et al. 2004), and type III intermediate filaments including vimentin, GFAP, and desmin (Goto et al. 2003; Kawajiri et al. 2003).
We previously detected protein kinase activity that phosphorylates GFAP, one of type III IF protein, specifically at the cleavage furrow from anaphase to cytokinesis (Nishizawa et al. 1991; Matsuoka et al. 1992). We tentatively named this kinase cleavage furrow (CF) kinase. At a later time, by several in vitro and in vivo studies, we revealed that Aurora-B and Rho-kinase/ROCK is the two CF kinases, and the substrates are several IF proteins including GFAP, desmin, and vimentin (Kosako et al. 1997, 1999; Yasui et al. 1998; Goto et al. 1998, 2003; Kawajiri et al. 2003). We have identified that vimentin is phosphorylated at Ser71 by Rho-kinase/ROCK and at Ser72 by Aurora-B, respectively (Goto et al. 1998, 2003). Vimentin phosphorylation at these sites occurs specifically at the cleavage furrow. Vimentin with mutations at Aurora-B or Rho-kinase/ROCK phosphorylated sites induce an aberrant long bridge-like IF structure (IF-bridge) between the unseparated daughter cells. Mutations at both Aurora-B and Rho-kinase/ROCK phosphorylated sites synergistically cause IF-bridge (Goto et al. 2003). Desmin is another common substrate of Aurora-B and Rho-kinase/ROCK. The synergistic IF-bridge emergence is also observed in desmin-mutated cells (Kawajiri et al. 2003). These evidences suggest that phosphorylation of IF proteins by Aurora-B and Rho-kinase/ROCK play pivotal roles in IF separation into two daughter cells, and it is significant to investigate if there is an interrelationship between these two CF kinases, Aurora-B and Rho-kinase/ROCK, during cytokinesis.
Here we present evidence that depletion of Aurora-B or Rho-kinase/ROCK does not affect each other's localization. Reduction of Aurora-B or Rho-kinase/ROCK does not change phosphorylation level of the other substrates at the cleavage furrow. ERM is not phosphorylated by Aurora-B nor Rho-kinase/ROCK, but by another undetermined protein kinase at the cleavage furrow.
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Results |
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In order to investigate if there is a relationship between Aurora-B and Rho-kinase/ROCK in cytokinesis, we depleted Aurora-B and/or Rho-kinase/ROCK by using small interfering RNA (siRNA) technique and analysed the effects to cells. Immunoblot indicates that every siRNA transfection down-regulated each target protein in HeLa cells lysates (Fig. 1A). We confirmed that Aurora-A was not down-regulated by Aurora-B siRNA (data not shown). We next performed immunocytochemistry to observe the effect of RNAi to localization of Aurora-B and Rho-kinase/ROCK. As shown in Fig. 1B, Aurora-B and Rho-kinase/ROCK2 accumulated at the spindle midzone and cleavage furrow in control HeLa cells (Fig. 1B,a–c). As well as immunoblot, it was indicated that every siRNA transfection abolished each target kinase in HeLa cells by immunostaining (Fig. 1B,d–l). We detected that although Aurora-B was exactly reduced by Aurora-B siRNA, the localization of Rho-kinase/ROCK at the cleavage furrow was not altered (Fig. 1B,d–f). Furthermore, when Rho-kinase/ROCK2 was reduced by Rho-kinase/ROCK1,2 siRNA, the localization of Aurora-B at the spindle midzone was not altered (Fig. 1B,g–i).
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Suppression of Aurora-B by siRNA or inhibitor causes multinucleation (Hauf et al. 2003). In a similar way, suppression of Rho or Rho-kinase/ROCK by using their inhibitor or dominant negative mutant also induces multinucleation (Kishi et al. 1993; Mabuchi et al. 1993; Yasui et al. 1998). But it remains unknown about the effects on cells by double depletion of Aurora-B and Rho-kinase/ROCK at the same time. So, we next analysed Aurora-B and/or Rho-kinase/ROCK depleted HeLa cells to observe the phenotype of cells when both kinases are suppressed. As shown in Fig. 2A, in contrast to mononucleate control HeLa cells (Fig. 2A,a,b), Aurora-B, Rho-kinase/ROCK, and both depletions induced multinucleation to HeLa cells (Fig. 2A,c–h). The frequency of multinucleate cell emergence by siRNA was counted. As a result, depletion of Aurora-B or Rho-kinase/ROCK induced multinucleation in 
35% or 15% cells, respectively, and additively 50% cells showed the multinucleate phenotype by depletion of both kinases (Fig. 2B).
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Vimentin is one of putative common substrates phosphorylated by Aurora-B and Rho-kinase/ROCK. Next, we investigated whether or not the phosphorylation of vimentin by Aurora-B are affected by Rho-kinase/ROCK depletion, and vice versa. We have elucidated that Aurora-B and Rho-kinase/ROCK phosphorylate vimentin at several sites, and produced site- and phosphorylated state-specific antibodies (Goto et al. 1998, 2003; Yasui et al. 2001; Fig. 3A,B). Immunocytochemistry indicates that vimentin Ser71 and Ser72 were phosphorylated at the cleavage furrow in control cells (Fig. 3C,a–d). Aurora-B depletion induced the reduction of cleavage furrow-specific phosphorylation of vimentin at Ser72 but not vimentin at Ser71 (Fig. 3C,e–h). On the other hand, Rho-kinase/ROCK depletion induced the reduction of cleavage furrow-specific phosphorylation of vimentin at Ser71 but not vimentin at Ser72 (Fig. 3C,i–l). Note that Aurora-B depletion did not affect phosphorylation of vimentin Ser71 by Rho-kinase/ROCK, and Rho-kinase/ROCK depletion did not affect phosphorylation of vimentin Ser72 by Aurora-B, at the cleavage furrow. (Fig. 3C,e,k)
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It is reported that ERM are phosphorylated by Rho-kinase/ROCK at carboxyterminal threonine (ezrin-Thr567, radixin-Thr564, and moesin-Thr558; Matsui et al. 1998). In this study, we confirmed that not Aurora-B but Rho-kinase/ROCK phosphorylates radixin in vitro (data not shown). However, cleavage furrow-specific phosphorylation of ERM was not changed by Aurora-B and/or Rho-kinase/ROCK depletion (Fig. 4f,i,l). We next investigated cleavage furrow-specific phosphorylation of ERM under the inhibition of Rho or Rho family proteins including Rho, Rac, Cdc42 by using C3 or toxin B, respectively. Although certain toxins-treated HeLa cells arrested cell cycle, phosphorylation of MLC and MBS at the cleavage furrow was reduced in the cells proceeding into cytokinesis (Fig. 4m,n,p,q). However, we found that cleavage furrow-specific ERM phosphorylation was not reduced by C3 or toxin B treatment (Fig. 4o,r).
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Discussion |
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Aurora-B and Rho-kinase/ROCK independently phosphorylate vimentin specifically at the cleavage furrow from anaphase to cytokinesis
We have developed site- and phosphorylation state-specific antibodies using synthesized phosphopeptides for the first time (Nishizawa et al. 1991; Matsuoka et al. 1992), and the method to produce the antibodies is used all over the world. Using the antibodies, we have indicated that IF proteins including vimentin, desmin, GFAP are putative common substrates for Aurora-B and Rho-kinase/ROCK at the cleavage furrow during cytokinesis (Kosako et al. 1997, 1999; Yasui et al. 1998; Goto et al. 1998, 2003; Kawajiri et al. 2003). In the present study, we showed that Aurora-B depletion abolished phosphorylation of vimentin at Ser72, but not Ser71. We also showed that Rho-kinase/ROCK depletion abolished phosphorylation of vimentin at Ser71, but not Ser72 (Fig. 3C). So, we confirm that vimentin is a common substrate for Aurora-B and Rho-kinase/ROCK, which phosphorylates vimentin at Ser72 and Ser71 at the cleavage furrow during cytokinesis, respectively. These findings suggest that Aurora-B and Rho-kinase/ROCK independently phosphorylate vimentin (Fig. 5).
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MLC is a component of actomyosin bundles. Phosphorylation of MLC induces actin–myosin interaction and thereby generates contractile force. Rho-kinase/ROCK is reported to phosphorylate MLC at Ser19 (Amano et al. 1996; Matsumura et al. 1998). It is also reported that MLC is phosphorylated at Ser19 by immunoprecipitated Aurora-B (Murata-Hori et al. 2000). In this study, we indicated that cleavage furrow specific phosphorylation of MLC was reduced by Rho-kinase/ROCK depletion (Fig. 4g), which is consistent with our previous data using inhibitor (Kosako et al. 2000). In contrast, Aurora-B depletion did not change MLC phosphorylation (Fig. 4d), although Murata-Hori et al. reported that Aurora-B phosphorylated MLC in vitro (Murata-Hori et al. 2000). These findings suggest that the main kinase phosphorylates MLC at Ser19 at the cleavage furrow is Rho-kinase/ROCK, and Aurora-B does not relate to MLC phosphorylation during cytokinesis (Fig. 5).
Dephosphorylation of MLC is catalysed by a myosin light chain phosphatase (MLCP) and induces relaxation of contractile force. Phosphorylation of MBS is reported to inactivate MLCP (Kimura et al. 1996). The major sites of MBS phosphorylation by Rho-kinase/ROCK are reported as Rat3 Ser854 (corresponds to human Ser852) and chicken Thr695 (corresponds to human Thr696), of which Ser852 is reported as the specific phosphorylated site by Rho-kinase/ROCK (Feng et al. 1999; Kawano et al. 1999). In this study, we indicated that cleavage furrow-specific phosphorylation of MBS at Ser852 was reduced by Rho-kinase/ROCK depletion but not Aurora-B depletion (Fig. 4e,h). These findings suggest that the main kinase phosphorylates MBS at Ser852 at the cleavage furrow is Rho-kinase/ROCK. Because the reduction of MBS phosphorylation by Rho-kinase/ROCK RNAi is not complete, it cannot be ruled out that the suppression of Rho-kinase/ROCK is incomplete or that another kinase phosphorylates MBS at Ser852. Even in that case, because treatments using C3 or toxin B abolished the phosphorylation of MBS, the main kinase phosphorylates MBS function at least in a Rho dependent manner (Fig. 4n,q).
From above findings concerning MLC and MBS, we presume that MLC is phosphorylated by not Aurora-B but by Rho-kinase/ROCK to generate contractile force, and MBS is also predominantly phosphorylated by Rho-kinase/ROCK to assist the phosphorylation of MLC through inactivating MLCP during cytokinesis. Therefore, contractile ring contraction is regulated effectively under Rho-kinase/ROCK pathway (Fig. 5). This aspect is congruent with the proposition of Kaibuchi and coworkers (Amano et al. 1996). Because depletion of Aurora-B does not change phosphorylation level of MLC and MBS with or without Rho-kinase/ROCK RNAi (compare Fig. 4a with d, g with j, b with e, and h with k), we conclude that Aurora-B is not involved in the pathway Rho-kinase/ROCK phosphorylates MLC and MBS in vivo.
In this study, we presented that depletion of Aurora-B and/or Rho-kinase/ROCK induced cell multinucleation and the frequency of multinucleate cell emergence by Aurora-B RNAi was more frequent than Rho-kinase/ROCK RNAi (Fig. 2). Why the difference of the frequency arises? Although it cannot be ruled out the capability that the suppression of Rho-kinase/ROCK RNAi is incomplete, these findings imply that the pathway through Rho-kinase/ROCK has collateral pathway compensates for deficiency of Rho-kinase/ROCK. Interestingly, O’Connell et al. (1999) have reported that C3 treatment inhibited the cleavage of poorly adherent HeLa cells, but did not inhibit firmly attached normal rat kidney cells. These observations suggest that cell separation is regulated not only by Rho dependent pathway but also by Rho independent pathway such an adhesion-dependent response. In the present study, we indicated that Rho-kinase/ROCK depleted HeLa cells which possessed multinuclei were only about 15% in contrast to that the Aurora-B depleted cells which possessed multinuclei were about 35%. These findings suggest that such adhesion-dependent cytokinesis exists to some extent even in HeLa cells, or another undetermined pathway which does not involve Rho-kinase/ROCK may exist.
From the present study, we may understand the relationship between Aurora-B and Rho-kinase/ROCK during cytokinesis as the following. We have presented that Aurora-B or Rho-kinase/ROCK depletion does not alter localization of the others and does not affect the phosphorylation of the other substrates. These evidences make us consider that Aurora-B and Rho-kinase/ROCK do not communicate with each other in the progression of cytokinesis. In contrast, the crosstalk between Aurora-B and Rho has been previously reported. MgcRacGAP is functionally converted to a RhoGAP through phosphorylation at Ser387 by Aurora-B during cytokinesis, and plays an essential role in the completion of cytokinesis as a RhoGAP at the late step of cytokinesis (Minoshima et al. 2003). In combining our findings and previous reports, we may indicate that Aurora-B and Rho-kinase/ROCK independently regulate the progression of cytokinesis by phosphorylates respective substrates, and subsequently crosstalk in the late stage of cytokinesis through MgcRacGAP to complete the cytokinesis. Because some Aurora-B depleted cells exited cytokinesis, the necessity of MgcRacGAP for completion of cytokinesis remains a matter of research.
Possibility of existence of the novel cleavage furrow kinase which phosphorylates ERM
ERM are considered to function as general cross-linkers between the plasma membrane and actin filaments (Tsukita et al. 1997; Bretscher 1999; Mangeat et al. 1999; Tsukita & Yonemura 1999). The amino-terminal of ERM associate with cell-surface glycoprotein and other integral membrane proteins, in contrast to the carboxy-terminal of ERM associate with actin filaments. ERM concentrate not only at microvilli, filopodia, ruffling membrane, cell-cell adhesion sites, but also at the cleavage furrow (Tsukita & Yonemura 1999). It is also reported that over-expression of their carboxy-terminal halves perturbs cytokinesis (Henry et al. 1995). These findings imply that ERM play some important roles in cytokinesis. Until now, the mechanism of ERM activation has been studied. Phosphorylated ERM are stabilized as activated form and function as cross-linkers between actin filament and plasma membrane to form microvilli (Matsui et al. 1999). Several protein kinases have been reported to phosphorylate carboxy-terminal of ERM threonine residue. Protein kinase C (PKC) 
phosphorylates ezrin at Thr567 (Ng et al. 2001), and PKC
phosphorylates moesin at Thr558 (Pietromonaco et al. 1998) in vitro, but it is unclear these phosphorylations occurs in vivo. Rho-kinase/ROCK also phosphorylates carboxy-terminal of ERM in vitro, but not in vivo according to the study using inhibitor (Matsui et al. 1998, 1999). It is also reported that ERM phosphorylation by Rho-kinase/ROCK may be involved in the formation of peripheral bundles according to the study using dominant negative mutant of Rho-kinase/ROCK (Takaishi et al. 2000). As above, the effect of Rho-kinase/ROCK inhibition in vivo remains unclear. The myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) as an effector of Cdc42 also phosphorylates ERM in vitro and is thought to be a candidate for the kinase phosphorylates ERM at filopodia (Nakamura et al. 2000). However, what kinase phosphorylates ERM at the cleavage furrow during cytokinesis remains to be elucidated. In this study, although radixin was phosphorylated by Rho-kinase/ROCK in vitro (data not shown) as reported (Matsui et al. 1998), we have examined that suppression of Aurora-B, Rho-kinase/ROCK, or both did not reduce phosphorylation of ERM at the cleavage furrow (Fig. 4f,i,l). Previous report has indicated that treatments by Rho-kinase/ROCK inhibitor do not reduce but increase cleavage furrow-specific ERM phosphorylation in U251 cells (Kosako et al. 2000). These observations are in contrast with our present results, which may reflect the difference between inhibitors and RNAi treatment. In either case, our findings indicate that neither Aurora-B nor Rho-kinase/ROCK phosphorylates ERM at the cleavage furrow. Moreover, neither C3 nor toxin B treatment abolished the cleavage furrow-specific phosphorylation of ERM, although only in cases in which the toxin-treated cells proceed to cytokinesis (Fig. 4o,r). These findings lead us to conjecture that ERM phosphorylation at the cleavage furrow does not depend on Rho family proteins, such as Rho, Rac, and Cdc42, and another undetermined protein kinase phosphorylates ERM may exist (Fig. 5).
In conclusion, we indicate that Aurora-B and Rho-kinase/ROCK function without communicating to each other at the cleavage furrow in the progression of cytokinesis, and subsequently their communication through MgcRacGAP may arise at the late step of cytokinesis. Rho-kinase/ROCK phosphorylates MLC and MBS to effectively induce contractile ring contraction; in contrast, the substrates for Aurora-B during cytokinesis are not yet well revealed. Using site- and phosphorylation state-specific antibody, we point out the possibility of the existence of a novel protein kinase which phosphorylates ERM specifically at the cleavage furrow, and the kinase may act in a Rho, Rac, Cdc42 independent manner. Identification of the protein kinase phosphorylates ERM will help us to elucidate the molecular mechanism of cleavage furrow ingression in cytokinesis. These issues will be resolved in the future works.
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Experimental procedures |
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HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). RNAi was performed using siRNA as previously described (Elbashir et al. 2001). The target sequences in the human Aurora-B and ROCK1 cDNAs have been reported (Chevrier et al. 2002; Hauf et al. 2003). The target sequence in the human ROCK2 cDNA is 5'-AAGGCATCGCAGAAGGTTTAT-3' (M. Amano, personal communication). Synthetic sense and anti-sense oligonucleotides (for Aurora-B were obtained from Dharmacon and those for ROCK1, 2 from Qiagen, respectively. Oligonucleotides were transfected using OligofectAMINETM (Invitrogen) according to the manufacturer's instructions. In brief, 2 x 104 cells were seeded in wells of a 6-well Plate 24 h before transfection. siRNA duplex and OligofectAMINETM were diluted in media, mixed, and incubated for 20 min. siRNA/lipid complexes were then added to cells and incubated for 4 h followed by addition of complete media. The cells were analysed 72 h after transfection. GST-C3 was produced and purified from Escherichia coli and GST was cut off with thrombin. HeLa cells were treated with 100 µg/mL C3 in DMEM (10% FCS) for 24 h or 50 ng/mL toxin B (CALBIOCHEM) in DMEM (10% FCS) for 10 h and thereby fixed to be analysed.
Immunoblotting
HeLa cells were lysed in Laemmli's sample buffer and boiled at 100 °C for 5 min. Lysates were then separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. The blots were incubated with anti-AIM1 (Aurora-B), anti-ROCK1, anti-ROCK2 (BD Transduction Laboratories), and anti--tubulin (SIGMA) mouse monoclonal antibody, and then with horseradish-peroxidase-conjugated second antibody. Immunoreactive bands were visualized using chemiluminescence detection reagents (Perkin-Elmer Life Sciences).
Preparation of proteins
Recombinant mouse vimentin was prepared from E. coli as previously described (Ogawara et al. 1995). GST-Aurora-B was purified from E. coli, as described (Goto et al. 2002). GST-Rho-kinase/ROCK-catalytic domain (GST-Rho-K-cat) was purified from Sf9 cells and kindly provided by Dr K. Kaibuchi (Amano et al. 1996).
Phosphorylation of vimentin
The phosphorylation reactions for GST-Aurora-B and GST-Rho-K-cat were performed as previously described (Goto et al. 1998, 2003). In brief, the phosphorylation reaction for vimentin was performed at 25 °C in 100 µL of 25 mM Tris-Cl (pH 7.5), 2 mM or 0.4 mM MgCl2, 100 µM[
-32P]-ATP, 0.1 µM calyculin A, and 150 µg/mL vimentin in the presence of either 10 µg/mL GST-Aurora-B or GST-Rho-K-cat. The reaction was stopped by the addition of Laemmli's sample buffer and boiled. Reacted vimentin were subjected to SDS-PAGE, visualized by autoradiogram, and immunoblotted with following antibodies: 4H4 (anti-mouse vimentin) mouse mAb diluted 1 : 50; GK71 (anti-phosphorylated vimentin-Ser71) rabbit pAb (Goto et al. 1998) diluted 1 : 500; YG72 (anti-phosphorylated vimentin-Ser72) rabbit pAb (Yasui et al. 2001) diluted 1 : 500 with 5% skim milk.
Immunofluorescence
HeLa cells were grown on coverslips, and then fixed by incubation with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min. For staining with anti-Rho-kinase/ROCK2 and CP-ERM, cells were fixed with ice-cold 10% trichloroacetic acid for 15 min as described (Hayashi et al. 1999). The fixed cells were permeabilized with PBS containing 0.1% Triton X-100 (except using methanol for GK71 and YG72 staining) for 10 min. Incubation with primary antibodies diluted in PBS containing 1% sucrose and 1% bovine serum albumin was for 1 h at room temperature. After three washes with PBS, cells were incubated for 1 h with appropriate secondary antibodies diluted 1 : 400 and subsequently washed with PBS. Then DNAs were stained with 0.5 µg/mL 4’6-diamideine-phenylindole-dihydrochloride (DAPI) or 0.5 µg/mL propidium iodide (PI) for 10 min at room temperature. The following antibodies were used for indirect immunofluorescence microscopy: anti-AIM1 (Aurora-B) mouse mAb (BD Transduction laboratories) diluted 1 : 100; anti-Rho-kinase/ROCK2 rabbit pAb (Kosako et al. 1999) diluted 1 : 450; anti--tubulin (Sigma) mouse mAb diluted 1 : 600; GK71 (anti-phosphorylated vimentin-Ser71) rabbit pAb (Goto et al. 1998) diluted 1 : 100; YG72 (anti-phosphorylated vimentin-Ser72) rabbit pAb (Yasui et al. 2001) diluted 1 : 100; pp2b (anti-phosphorylated MLC-Ser19) rabbit pAb (Matsumura et al. 1998) diluted 1 : 100; pS854 (anti-phosphorylated Rat3 MBS-Ser854 correspond to human MBS-Ser852) rabbit pAb (Kawano et al. 1999) diluted 1 : 10; CP-ERM (anti-phosphorylated ezrin-Thr567, radixin-Thr564, and moesin-Thr558) rat mAb (Matsui et al. 1998) diluted 1 : 5; Alexa Fluor 488-conjugated goat anti-rabbit, anti-rat, anti-mouse IgG (Molecular Probe) and Cy3-conjugated anti-mouse IgG (Amersham Biosciences) diluted 1 : 400. Fluorescently labelled cells were examined using an Olympus BX60F5 microscope.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: minagaki{at}aichi-cc.jp
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References |
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Received: 4 October 2004
Accepted: 15 November 2004
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