Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.
- contractile ring
- fluorescence recovery after photobleaching (FRAP)
- myosin regulatory light chain (MRLC)
During cytokinesis in animal cells, a CR (contractile ring) consisting of myosin II and F-actin (filamentous actin) bundles is formed at the equator of the cell [1,2]. In analogy to muscle, it is believed that the contractile force of the ring is generated by the interaction between myosin II and F-actin [2–5]. Although the CR has been examined by electron microscopy in a variety of species, including newts, cultured vertebrate cells and fission yeast [6–8], specific contractile structures such as the sarcomere have not been reproducibly observed. Thus the precise mechanism by which the CR achieves constriction is still not understood.
Myosin II ATPase activity is stimulated by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. Whereas mono-phosphorylation of MRLC at Ser19 increases both the actin-activated Mg-ATPase activity and the assembly of myosin II filaments, diphosphorylation of MRLC at Thr18/Ser19 results in higher ATPase activity and assembly of myosin II  and thick actin bundles . It has also been shown that both mono- and di-phosphorylated MRLC (pMRLC) were localized in the CR during cytokinesis [10–14].
Recent studies have demonstrated the roles of pMRLC in each step of cell division. Live-cell imaging studies revealed that phosphomimic MRLCs are recruited and retained in the CR of HeLa and Drosophila S2 cells [15,16]. In addition, myosin II Mg-ATPase activity is not required for the recruitment of myosin II [15,17,18] and F-actin [19,20] to the furrow in dividing cultured mammalian cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC (AA-MRLC), in which Thr18 and Ser19 are each replaced by alanine, show a slower furrowing, although the amount of myosin II and F-actin in the CR does not fluctuate compared with the levels in the control cells . Furthermore, the slower furrowing induced by a Rho-kinase inhibitor, Y-27632, was rescued by phosphomimic MRLCs. These accumulating results suggest that myosin II activity enhances the progress of furrow ingression, but not the recruitment of myosin II and F-actin along the CR of dividing cells. However, little information is available to understand how pMRLC regulates myosin II and F-actin to control the speed of furrow ingression during cytokinesis.
The present study aimed to clarify how pMRLC controls furrow ingression in cultured mammalian cells. To investigate this, we measured the dynamic turnover of myosin II and actin in the CR using FRAP (fluorescence recovery after photobleaching) during cytokinesis in HeLa cells expressing EGFP- (enhanced green fluorescent protein) or mCherry-tagged recombinant actin and MRLCs including AA-MRLC and phosphomimic MRLC. We demonstrate that the inhibition of myosin II ATPase activity by a myosin II inhibitor, blebbistatin, caused a delay in the furrowing speed and enhanced the turnover of MRLC and actin in the CR. In addition, AA-MRLC, but not other MRLCs, and the inhibition of Rho-kinase by Y-27632 also accelerated the turnover of MRLC and actin in the CR. The effect of Y-27632 was cancelled in the cells expressing phosphomimic MRLCs. Taken together, the enhancement of myosin II and actin turnover in the CR induces slower furrowing in dividing HeLa cells, suggesting that the phosphorylation state of MRLC in the CR plays a crucial role in the control of furrow ingression during cytokinesis.
MATERIALS AND METHODS
HeLa cells (RCB0007) were obtained from RIKEN Cell Bank (Tsukuba, Japan). The cells were cultured in Eagle's minimal essential medium (Nissui Pharmaceutical) supplemented with 10% (v/v) fetal bovine serum (Biological Industries) at 37°C with 5% CO2.
Plasmid construction and transfection
The plasmids generated by the subcloning of Wt- (wild-type), AA-, AD- (T18A/S19D double mutant) and DD- (T18D/S19D double mutant) MRLC2 into the pEGFP-N1 vector (Clontech) were prepared as described previously . The expression vectors for mCherry–actin and Wt-MRLC–mCherry were generated by fusing human β-actin and MRLC2 cDNAs respectively to mCherry cDNA . The construct for expression of mCherry–MHC-IIA (myosin heavy chain IIA) was generated by replacing the TRE (tetracycline-response element)–EGFP (enhanced green fluorescent protein) in pTRE-GFP-NMHC-IIA with CMV (cytomegalovirus)–mCherry. The transfection of HeLa cells was carried out using Lipofectamine™ (Invitrogen) following the manufacturer's instructions.
FRAP analysis and drug treatment
For FRAP experiments, HeLa cells were plated on a glass-based dish (Asahi Glass). At 2 days after transfection, cells were observed using a FV1000-D confocal laser-scanning microscope (Olympus) with a UPLSAPO 100× NA (numerical aperture) 1.40 oil-immersion lens (Olympus) in a stage top incubator (Tokai Hit) maintained at 37°C with 5% CO2. A single z-section was imaged and then an arbitrary region was photobleached with a 473 nm laser (100% power) for 2 s using a SIM scanner (Olympus). After photobleaching, images were acquired approximately every 5 s for 130 s for mitotic cells and every 1 min for 30 min for interphase cells. For photobleaching in the presence of a Rho-kinase inhibitor Y-27632 (kindly provided by Welfide Corporation, Osaka, Japan; now obtained from Sigma–Aldrich) and imaging were performed as described above. For photobleaching in the presence of (S)-(−)-blebbistatin (Toronto Research Chemicals), cells were imaged and photobleached using a 559 nm laser. For quantitative analyses, the fluorescence intensity at the photobleached region was analysed using FluoView software (Olympus). The fluorescence intensity was normalized to the decrease in fluorescence after photobleaching. The FRAP results were fitted to the single-exponential equation R=P(1−exp−kt) using ImageJ 1.42, where R is the relative fluorescence intensity, P is the plateau value, k is the association constant and t is the time, then the t½ (half-time of recovery)=−ln(1/2)/k .
In our previous study , cells expressing AA-MRLC showed a slower furrowing. These results suggest that myosin II ATPase activity is required to control the speed of furrowing during cytokinesis in mammalian cells. First, we conducted FRAP experiments to determine whether the behaviour of the MRLC reflects the dynamics of myosin heavy chain itself by co-transfection with mCherry–MHC-IIA plasmids (Figures 1A–1E). As shown in Figure 1(A), we photobleached a region which included the CR and midzone (magenta rectangle) during time-lapse imaging, and measured the fluorescence intensity at the CR (blue rectangle) in each image to avoid overestimating diffused fluorescence at the midzone. Our results revealed that the exogenous MRLC has the same rate of exchange on the exogenous myosin heavy chain at the CR (Figures 1B and 1C, mitotic cells) and in stress fibres (Figures 1D and 1E, interphase cells), suggesting that the behaviour of exogenous MRLC reflects the dynamics of myosin II itself, in agreement with the previous results that the exogenous MRLC can access endogenous myosin heavy chain in cells . Next, we performed FRAP experiments in the presence of blebbistatin (100 μM) using confocal microscope to examine whether myosin II ATPase activity is required to regulate the turnover of myosin II and actin at the CR in HeLa cells (Figures 1F–1I). To eliminate phototoxicity and photoinactivation of blebbistatin after exposure to blue light [26,27], we generated plasmids encoding mCherry-tagged Wt-MRLC and actin, then transfected them into HeLa cells. Immunofluorescence revealed that Wt-MRLC–mCherry co-localized with endogenous myosin II in both interphasic and mitotic cells (see Supplementary Figure S1A at http://www.BiochemJ.org/bj/435/bj4350569add.htm) and mCherry–actin appeared to be incorporated into actin-containing structures, such as stress fibres and CR (Supplementary Figure S1B). In Wt-MRLC–mCherry-expressing cells (Figure 1F), the recovery t½ of Wt-MRLC was reduced by blebbistatin treatment (Figure 1G, left-hand panel). As shown in Figure 1(G) (right-hand panel), blebbistatin caused significant delays in the progression of furrowing during FRAP experiments. The effects of blebbistatin both on the Wt-MRLC turnover and furrow ingression did not depend on the incubation period at least up to 2 h (Supplementary Figure S1C). Likewise, in mCherry–actin-expressing cells (Figure 1H), the blebbistatin treatment caused not only the acceleration of actin turnover, but also a delay in furrow ingression (Figure 1H and Supplementary Figure S1D). In addition, we confirmed that the accumulation level of fluorescent proteins at the CR had no apparent effect on these results in the presence of blebbistatin (see Supplementary Figure S2A at http://www.BiochemJ.org/bj/435/bj4350569add.htm). Taken together, these results indicate that the inhibition of myosin II ATPase activity by blebbistatin reduces the speed of furrow ingression and enhances the turnover of myosin II and actin at the CR in dividing HeLa cells.
Myosin II consisting of either phosphorylated or phosphomimic MRLC is known to enhance ATPase activity [9,28,29]. Bresnick et al.  have also reported that myosin II consisting of AA-MRLC shows decreased actin-activated Mg-ATPase activity in vitro. These results indicated that pMRLC plays an important role in regulating the Mg-ATPase activity of myosin II in the cells. To determine the role of pMRLC in the dynamics of myosin II in HeLa cells, we performed FRAP experiments in cells expressing each phosphomimic MRLC–EGFP [15,22,25]. An arbitrary region in the stress fibres was photobleached (Figure 2A) and the fluorescence recovery in the region was analysed (Figure 2B). As shown in Figure 2(C) and Supplementary Table S1 (at http://www.BiochemJ.org/bj/435/bj4350569add.htm), AA-MRLC exhibited the fastest recovery (t1/2=322±45.7 seconds) among others including Wt-, AD- and DD-MRLC (825±105, 498±37.7 and 650±119 s respectively). We also analysed the dynamics of each MRLC–EGFP in cells co-expressing mCherry–actin (Supplementary Table S1). Again, the fluorescence of AA-MRLC recovered most rapidly (t1/2=215±37.6 s). Wt-, AD- and DD-MRLC recovered with t1/2=930±230, 773±249 and 726±276 s respectively. These results suggest that non-phosphorylatable MRLC accelerates the turnover of myosin II in stress fibres of HeLa cells, in agreement with a previous study in another type of epithelial cell [MDCK (Madin–Darby canine kidney) cells] stably expressing each MRLC–EGFP mutant .
To examine the function of pMRLC on the dynamic behaviour of myosin II and actin at the CR, we performed FRAP experiments in dividing cells co-expressing each MRLC–EGFP mutant and mCherry–actin (Figures 3A–3D). As observed in interphase stress fibres, AA-MRLC at the CR recovered more rapidly (t1/2=55.8±4.3 s) than Wt-, AD- and DD-MRLC (t1/2=89.2±11.3, 78.5±5.4 and 180±24 s respectively) (Figure 3E). The mobility of mCherry–actin was also affected by the co-expression of MRLC–EGFP mutants. Concomitantly with the behaviour of mutant MRLCs, mCherry–actin recovered more rapidly in AA-MRLC-expressing cells (t1/2=33.9±2.0 s) than in cells expressing Wt-, AD- or DD-MRLC (57.7±9.9, 54.7±5.4 and 68.2±7.0 s respectively) (Figure 3F). To determine the possibility that the expression level of fluorescent proteins influences the turnover of MRLC and actin, we analysed the correlation between the accumulation level of fluorescent proteins at the CR and the t1/2 in each cells (Supplementary Figure S2B). Thus we confirmed that the amount of accumulation of fluorescent proteins at the CR has no effect on the turnover of MRLC and actin.
Our previous study demonstrated that Y-27632 disturbs the localization of monophosphorylated MRLC, but not F-actin, along the CR in dividing HeLa cells . In addition, it has also been elucidated that Y-27632 causes a delay in furrowing in non-transfected cells  as well as in cells expressing MRLC mutants with the exception of phosphomimic MRLC . To determine further how the phosphorylation of MRLC controls the turnover of myosin II and actin at the CR, we examined whether the inhibition of Rho-kinase by Y-27632 has an effect on the turnover of MRLC and actin (Figures 4A–4D). In Wt-MRLC–EGFP- and mCherry–actin-co-expressing cells, Y-27632 significantly accelerated the turnover of MRLC and actin. The recovery t1/2 of Wt-MRLC and actin in the cells treated with Y-27632 decreased to levels similar to that of AA-MRLC and actin in AA-MRLC-expressing cells treated with or without Y-27632 (Figure 4E). In contrast, Y-27632 showed no effect on the turnover of MRLC and actin in cells expressing AD- and DD-MRLC, indicating that these phosphomimic MRLCs cancel the acceleration of turnover of MRLC and actin in the CR caused by Y-27632. Taken together, these results suggest that non-phosphorylated form of MRLC increases the turnover of both myosin II and actin in the CR of dividing HeLa cells.
It is well known that MRLC is phosphorylated by several kinases such as Rho-kinase [13,32], Citron kinase , ZIP-kinase (zipper-interacting protein kinase) , MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase)  and MLCK (myosin light chain kinase) . Recent reports suggest that, in mammalian cultured cells, Rho signalling plays an important role in assembling and maintaining a CR . In particular, Zhao and Fang  and Kamijo et al.  have shown that the localization of pMRLC at the CR is regulated by Rho signalling. In addition, the inhibition of MLCK by ML-7 and BATI-peptide has no effect on cytokinesis in mammalian cultured cells (S. Nazumi, Y. Akioka, K. Hamao and H. Hosoya, unpublished work). These results suggest that Rho-dependent kinases including Rho-kinase and Citron kinase, but not other kinases play major roles to phosphorylate MRLC during cytokinesis. Citron kinase phosphorylates MRLC directly at Thr18/Ser19 , whereas Rho-kinase phosphorylates at Thr18/Ser19  or only Ser19 . Thus, in the present study, to inhibit the MRLC phosphorylation at Ser19 or Thr18/Ser19 by Rho-dependent kinases at the CR, Rho-kinase inhibitor (Y-27632) and AA-MRLC were used for FRAP experiments.
In dividing HeLa cells expressing non-phosphorylatable MRLC, we observed the enhancement of MRLC and actin turnover at the CR (the present study) and slower furrowing . Our results also revealed that suppression of myosin II activity by blebbistatin induces the enhancement of MRLC and actin turnover at the CR and slower furrowing. These results suggest that myosin II activity plays a crucial role in the control of the speed of furrow ingression and myosin II/actin dynamics at the CR during cytokinesis.
Although it is generally believed that blebbistatin is an inhibitor of cytokinesis, we demonstrated that blebbistatin (100 μM) induced a delay in furrow ingression rather than complete inhibition of cytokinesis under our experimental conditions (Figure 1). A recent study claimed that blebbistatin inhibited cytokinesis in an epithelial cell line stably expressing GFP (green fluorescent protein)–actin, and the furrowing of a dividing cell was apparently blocked 15 min after the addition of 75 μM blebbistatin . However, as no quantitative results showing the inhibition of cytokinesis by blebbistatin are available, it is difficult to conclude that blebbistatin inhibits cytokinesis. Straight et al.  also reported that addition of blebbistatin to dividing HeLa cells blocked furrow ingression. However, less than 30% of cells showed binucleation even in the presence of 100 or 150 μM blebbistatin. In other words, more than 70% of cells successfully divided, suggesting that blebbistatin exhibits a delay in, but not inhibition of, furrowing, which is consistent with our results in HeLa cells. Thus these results confirm that blebbistatin induces a delay in furrowing during cytokinesis.
Our observation that blebbistatin treatment enhances the turnover of actin at the CR is somewhat surprising, as two independent studies have reported that actin turnover in the equatorial region of dividing cells was reduced by blebbistatin [19,20]. Although Murthy and Wadsworth  used GFP–actin in their FRAP experiments, previous reports indicated that blebbistatin is blue-light-sensitive [26,27]. Therefore the observation based on GFP fluorescence does not eliminate the possible toxic effects of blebbistatin after exposure to blue light on cell metabolism. Thus mCherry–actin was selected for our FRAP experiments. In the second study, rhodamine-labelled actin was microinjected during prometaphase and bleached at the approximate onset of anaphase . Several reports have revealed that 20–30 min after microinjection, most stress fibres in interphase cells were uniformly labelled by fluorescently labelled actin [39–41]. However, in M-phase, the fluorescence of rhodamine-labelled actin, which was microinjected during metaphase, was not evenly distributed throughout the cytoplasm of the dividing cell even at telophase . This prompted us to adopt the transfection with GFP- or mCherry-tagged proteins, but not the microinjection of labelled proteins, especially in mitotic cells, for the FRAP experiments. Thus our FRAP experiments using transfection with mCherry–actin/MRLC is more preferable to previous studies using other probes for the examination of myosin II and actin turnover at the CR during cytokinesis. The results obtained from our FRAP experiments in the presence of blebbistatin were supported by those from our other experiments in cells transfected with AA-MRLC and treated with Y-27632.
In the present study, our FRAP experiments showed that the exchange rate of fluorescent MRLC and/or actin between the contractile apparatus and its pool in cytoplasm is faster in mitotic than in interphasic cells (Table 1a). Attractive factors causing this difference of myosin II/actin dynamics might be myosin II/actin-interacting proteins such as caldesmon. It has been reported that caldesmon binds with myosin II and actin, blocks the interaction of myosin II with actin and then inhibits actin-activated myosin II ATPase activity [43,44]. Interestingly, several reports revealed that mitosis-specific phosphorylation of caldesmon by Cdc2 kinase reduced its actin-binding activity [45,46]. Additional experiments showed that caldesmon localizes at stress fibres in interphase, but in the cytoplasm during mitosis . These results suggest that unphosphorylated caldesmon inhibits myosin II/actin dynamics by binding to actomyosin at stress fibres in interphase cells, and phosphorylated caldesmon no longer binds to actomyosin and enhances myosin II/actin dynamics at the CR in mitotic cells. To confirm this, FRAP experiments in cells transfected with phosphomimic or unphosphorylatable form of caldesmon are required.
How does pMRLC regulate the dynamics of actin to control furrow ingression? Previously, we observed that the overexpression of DD-MRLC, but not AA-MRLC, organized a large amount of thick actin bundles in interphase cells . We revealed further that the overexpression of either ZIP-kinase or Rho-kinase, which efficiently diphosphorylates MRLC, induced the formation of striking aggregates and bundles of F-actin in HeLa cells [13,48]. In addition, it has been reported that the phosphorylation of MRLC induced by lysophosphatidic acid caused the formation of stress fibres in interphase cells . These results suggest that myosin II, including pMRLC, organizes F-actin into thick actin bundles in cells. Our previous study revealed that the amount of myosin II and F-actin at the CR was not dependent on the phosphorylation state of MRLCs . It should be emphasized that the organization of F-actin into thick actin bundles at the CR may be dependent on only on the phosphorylation state of MRLC, but not the total amount of myosin II and F-actin at the CR. These results allow us to speculate a hypothesis for the control of furrow ingression in dividing cells (Table 1b). Phosphorylation of MRLC is required to organize F-actin into thick actin bundles at the CR, which induces the stabilization of myosin II and F-actin at the CR. Then, furrow ingression will be faster during cytokinesis. In contrast, AA-MRLC or Y-27632/blebbistatin reorganizes thick actin bundles into F-actin, inducing a rapid turnover of myosin II and actin at the CR. The presence of unbundled F-actin at the CR would induce slower furrow ingression. These results suggest that the rate of myosin II/actin turnover at the CR regulates the speed of furrow ingression. Further electron microscopic observation is required to examine the structure of F-actin at the CR in cells transfected with phosphomimic or non-phosphorylatable MRLC.
Tomo Kondo performed all of the experiments except for the generation of Wt-MRLC–mCherry (by Keiju Kamijo) and mCherry–MHC-IIA (by Masayuki Takahashi). Hiroshi Kimura gave technical suggestions for the mathematical analysis of FRAP results to Tomo Kondo and Hiroshi Hosoya. Makiko Morita helped with the analysis of FRAP results. Tomo Kondo, Kozue Hamao and Horishi Hosoya discussed and wrote the paper.
This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan to H.H.
We thank Dr Keigi Fujiwara (University of Rochester, Rochester, NY, U.S.A.) for anti-MHC-IIA, Dr Roger Y. Tsien (University of California, San Diego, CA, U.S.A.) for mCherry cDNA, Dr Robert S. Adelstein (National Heart, Lung and Blood Institute, Bethesda, MD, U.S.A.) for pTRE-GFP-NMHC-IIA and Dr Naoki Watanabe (Tohoku University, Sendai, Japan) for helpful comments.
Abbreviations: CR, contractile ring; EGFP, enhanced green fluorescent protein; F-actin, filamentous actin; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; MHC-IIA, myosin heavy chain IIA; MLCK, myosin light chain kinase; MRLC, myosin II regulatory light chain; pMRLC, phosphorylated MRLC; Wt, wild-type; ZIP-kinase, zipper-interacting protein kinase
- © The Authors Journal compilation © 2011 Biochemical Society