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Genes to Cells (2009) 14, 555-568. doi:10.1111/j.1365-2443.2009.01288.x
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Direct evidence for roles of phosphorylated regulatory light chain of myosin II in furrow ingression during cytokinesis in HeLa cells

 Satoshi Asano, Kozue Hamao and Hiroshi Hosoya*

Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Phosphorylation of myosin II is thought to play an important role in cytokinesis. Although it is well known that phosphorylated regulatory light chain of myosin II (P-MRLC) localizes along the contractile ring, it is not clear how P-MRLC controls myosin II and F-actin in furrow ingression during cytokinesis. To elucidate roles of P-MRLC in furrow ingression, HeLa cells transfected with EGFP-tagged wild-type or each MRLC mutant were observed using a live-imaging microscope. Time-lapse observation revealed that a delay of furrow ingression was observed in the nonphosphorylatable form of MRLC (AA-MRLC)-expressing cell but not in the wild-type or phospho-mimic MRLC-expressing cell. Among each form of MRLC-expressing cell, the total amount of P-MRLC including phospho-mimic MRLCs was smallest in the cell expressing AA-MRLC. However, the amount of F-actin and myosin II at the contractile ring in the AA-MRLC-expressing cell was the same as that in the normal cell. Interestingly, delay of furrow ingression by a Rho-kinase inhibitor, Y27632, was rescued by phospho-mimic MRLCs. These results suggest that the P-MRLC is essential for the progress of furrow ingression but not the retainment of F-actin and myosin II in the contractile ring of dividing HeLa cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Myosin II is required to regulate cell motility (Berlot et al. 1985) and cytokinesis (Mabuchi & Okuno 1977; De Lozanne & Spudich 1987). In animal cells, cytokinesis involves an actomyosin-based contractile ring that forms and constricts at the division plane. Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin essential light chains (MELC), and two myosin regulatory light chains (MRLC) (Sellers 2000). In the vertebrate smooth muscle and nonmuscle myosin II, it is believed that the phosphorylation of MRLC determines the ability of MHC to bind to and move along actin filaments (Sellers 1999). While the mono-phosphorylation of MRLC at Ser-19 can activate the ATPase activity of myosin II and support actin motility in vitro, di-phosphorylation of MRLC at Ser-19 and Thr-18 results in several fold higher ATPase activity (Ikebe & Hartshorne 1985), thick actin filament assembly (Murata-Hori et al. 2001; Ueda et al. 2002) and enhanced tension of stress fibers in the cell (Mizutani et al. 2006), suggesting a possible role for bundling and constriction of actin filaments along contractile ring in dividing cells. Interestingly, in Dictyostelium cells, phosphorylation of MHC but not MRLC regulates assembly of myosin II. When the tail of MHC was phosphorylated at three specific threonine residues, the tail bends and the myosin II filament disassembles into monomers (Egelhoff et al. 1993). Thus, phosphorylation on both MRLC and MHC is important to regulate the function of nonmuscle myosin II.

DeBiasio et al. (1996) have reported that myosin fibers flow toward the cleavage furrow of mitotic cell during anaphase and form a meshwork. This cortical flow of myosin at the furrow has been widely observed in Dictyostelium cells (Yumura & Uyeda 1997), Xenopus eggs (Noguchi & Mabuchi 2001) and fission yeast (Pelham & Chang 2002). Immunostaining (Straight et al. 2003) and live-imaging (Guha et al. 2005; Miyauchi et al. 2006) studies using blebbistatin, an inhibitor of myosin ATPase activity, reported that recruitment of myosin II to the furrow does not require its ATPase activity in mammalian cultured cells. In Dictyostelium cells, fluorescence recovery after photobleaching experiments showed that phosphorylation of MHC is responsible for the turnover of myosin II in the furrow of dividing cells (Yumura 2001). In addition, recent total internal reflection fluorescence microscopic observation revealed that motorless myosin II filaments accumulate into the furrow in Dictyostelium cells independently of cortical flow (Yumura et al. 2008). Thus, growing evidence suggests that myosin II activity is not essential for recruitment of myosin II to the contractile ring before furrow ingression.

Previous immunostaining studies have shown that mono- and di-phosphorylated MRLCs are highly enriched at the cleavage furrow of dividing mammalian cultured cells (Matsumura et al. 1998; Murata-Hori et al. 1998; Iwasaki et al. 2001; Matsumura et al. 2001; Gerashchenko et al. 2002). Several studies using recombinant phospho-mimic MRLCs in HeLa cells and Drosophila S2 cells also confirmed the previous results obtained from immunostaining studies (Iwasaki et al. 2001; Miyauchi et al. 2006; Dean & Spudich 2006). Recent live-imaging studies revealed that phospho-mimic MRLCs were recruited to and retained in the cleavage furrow in HeLa cells and Drosophila S2 cells (Miyauchi et al. 2006; Dean & Spudich 2006). In HeLa cells, recruitment but not retainment of nonphosphorylatable form of MRLC into the furrow was also observed by live-imaging (Miyauchi et al. 2006). It has been reported that the phosphorylation of MRLC might induce an increase in the rate of assembly or cortical association of myosin II in the contractile ring during furrow ingression (Uyeda et al. 2004; Eggert et al. 2006). These results suggest that the phosphorylation of MRLC plays an important role to retain myosin II in the cleavage furrow.

How does P-MRLC in the cleavage furrow control furrow ingression? This question is an unsolved problem. Our studies aimed to clarify the role of P-MRLC during furrow ingression in mammalian cultured cells. To investigate the role of P-MRLC, four types of enhanced green fluorescent protein (EGFP)-tagged recombinant MRLCs, wild-type, nonphosphorylatable form (AA-MRLC), and mono- and di-phospho-mimic MRLC (AD-MRLC and DD-MRLC, respectively), were designed as previously described (Uchimura et al. 2002) and transfected into HeLa cells. Using real-time imaging of EGFP-tagged MRLCs, we show that P-MRLC plays important roles to control the speed of furrow ingression in dividing HeLa cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Delay of furrow ingression in the cell transfected with AA-MRLC

To determine roles of P-MRLC during furrow ingression, we have transiently over-expressed MRLC-EGFP or each MRLC mutant in HeLa cells. The progress of furrow ingression was shown using relative value of diameter at the equatorial region of dividing cells in each time from anaphase onset to late cytokinesis divided by that in start cytokinesis. As shown in Supporting Fig. S1, cells transfected with four kinds of MRLC-EGFP, which a little bit elongated along the chromosome-to-chromosome axis, showed more than 1 in values of relative diameter of dividing cells after anaphase onset. When the cell started furrowing (start cytokinesis), the value of relative diameter of dividing cells was designated as 1 (Supporting Fig. S1B). All of these transfected cells showed the same cell division time from anaphase onset to start cytokinesis (Table 1). Next, the progression of furrow ingression was re-plotted using relative value of diameter from start cytokinesis to late cytokinesis (Fig. 1B). Although four kinds of MRLC-EGFP were concentrated into the contractile ring region of dividing cells from start cytokinesis to late cytokinesis, DD-MRLC is more preferentially localized to the cell cortex and the furrow (Fig. 1A), suggesting that the phosphorylation status of MRLC affects cellular distribution of MRLCs. The interval between anaphase onset and the start cytokinesis was not shortened when DD-MRLC was expressed (Supporting Fig. S1), indicating that all needed for efficient cytokinesis is active myosin, and it is not important to switch-on the activity of myosin at an appropriate time point during cytokinesis. A similar conjecture has been put forward by Drosophila cells (Dean & Spudich 2006) and Dictyostelium cells (Uyeda & Spudich 1993). Interestingly, furrow ingression of AA-MRLC-expressing cells was slower than that of Wt- and phospho-mimic (DD- and AD-) MRLC-expressing cells (Fig. 1B and Table 1). These results revealed that phosphorylation of MRLC plays an important role after but not before start cytokinesis.


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  		Table 1  Cell division time from anaphase onset to 50% cytokinesis
 

Figure 1
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  		Figure 1  The progress of furrow ingression in HeLa cells expressed with each MRLC mutant. (A) Time lapse series of fluorescence images by conventional microscope in mitotic HeLa cells over-expressing EGFP-tagged Wt-, DD-, AD- or AA-MRLC during furrow ingression. Images were observed at 1 min intervals and shown for every 2 min. Wt-MRLC-transfected cells from start cytokinesis (a) to late cytokinesis (c) are shown. Bar, 10 µm. (B) The progress of furrow ingression in each MRLC mutant expressing cells from start cytokinesis to late cytokinesis. The width of cleavage furrow in HeLa cells transfected with Wt-, DD-, AD- or AA-MRLC was observed at each time point (the line drawn in Fig. 1A, panel b, for example), divided by that at start cytokinesis (the line drawn in Fig. 1A, panel a) and represented as relative diameter (ordinate). Abscissa shows minutes after start cytokinesis. The progress of furrow ingression is shown as decreasing of relative diameter. The result is shown as an average of at least 10 cells.

 
Amount of F-actin localized along the contractile ring in each MRLC-transfected cell

To elucidate how furrow ingression prolonged in the AA- but not other MRLC-expressing cells, the amount of F-actin in the contractile ring was examined during furrow ingression. In cells transfected with four kinds of MRLC-EGFP, fluorescence intensity of F-actin in the contractile ring was quantified (Fig. 2C). As shown in Fig. 2C (graph a), F-actin was gradually concentrated into the contractile ring region accompanying the progress of furrow ingression in the cells transfected with EGFP or four kinds of MRLC-EGFP. However, in all kinds of transfected cell, the average amount of F-actin from anaphase onset to late cytokinesis in the contractile ring region was the same (Fig. 2C, graph b). These results suggest the following: (i) the accumulation of F-actin into the furrow is unrelated to the phosphorylation state of MRLC in the contractile ring and (ii) the delay of furrow ingression in the cells expressing AA-MRLC is not dependent of the amount of F-actin in the contractile ring.


Figure 2
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  		Figure 2  Fluorescence intensity of F-actin in cells expressing EGFP, Wt-MRLC or each MRLC mutant during furrow ingression. (A) Localization of F-actin in HeLa cells expressing each MRLC mutant. Cells were fixed with 3.7% formaldehyde and processed for confocal microscopy to detect EGFP for MRLC mutant and TRITC fluorescence for F-actin. Bar, 10 µm. (B) Schematic diagram of dividing cells are shown. The major axis of the cell is defined as L; its cleavage furrow width is defined as d in (a). The contour of the contractile ring region is outlined in (b). (C) The amount of F-actin at the contractile ring region (B, panel b) in each transfected cell. Graph (a) shows fluorescence intensity of TRITC-phalloidin on the contractile ring region in the progress of furrow ingression. The abscissa of the graph was defined as d/L in schematic diagram in (B). Graph (b) shows the mean of fluorescence intensity of TRITC-phalloidin in the contractile ring region. Values represent standard error for at least 30 cells.

 
Amount of P-MRLC localized along the contractile ring in each MRLC-transfected cell

It is well known that P-MRLC is accumulated into the contractile ring. Here, the localization of P-MRLC in each MRLC-transfected cell was elucidated. Interestingly, in interphase cells (Fig. 3A), P-MRLC staining was observed only in EGFP- and Wt-MRLC-expressing cells but not DD-, AD- and AA-MRLC-expressing cells (marked with white asterisk). In mitotic cells, P-MRLC was also observed mainly along the cell cortex and the contractile ring region in EGFP- and Wt-MRLC-expressing cells but not in DD-, AD- and AA-MRLC-expressing cells (Fig. 3B). Based on the fluorescent images of Fig. 3B, the fluorescence intensity of P-MRLC both in the entire cell region and in the contractile ring region was determined (Fig. 3C,D). In DD-, AD- and AA-MRLC-expressing cells, the relative fluorescence intensity of P-MRLC in both the entire cell region and the contractile ring region was less than half of that in EGFP- and Wt-MRLC-expressing cells. Next, Western blotting analysis revealed that the amount of phosphorylated endogenous MRLC in four kinds of MRLC-EGFP-transfected cells decreased compared to that in EGFP-expressing cells (lower row in panel E). Exogenous Wt-MRLC but not DD-, AD- and AA-MRLC was phosphorylated (middle row in panel E), because Wt-MRLC but not other the three MRLC mutants has phosphorylatable sites (Thr-18 and Ser-19) by endogenous myosin kinases in the cell. As shown in Fig. 3F, in EGFP and four kinds of MRLC-expressing cells, the total amount of endogenous MRLC did not change. These results suggest that the expression of exogenous MRLC reduced the amount of endogenous P-MRLC but not expression of endogenous total MRLC. Taken together, in Wt-MRLC-expressing cells, phosphorylated exogenous Wt-MRLC might compensate the decrease of endogenous P-MRLC, inducing the obvious staining of P-MRLC in the cell. On the contrary, in other MRLC-expressing cells, the decrease of P-MRLC could be not compensated, showing no obvious staining of P-MRLC in each cell.


Figure 3
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  		Figure 3  The amount of P-MRLC in HeLa cells expressing EGFP, Wt-MRLC or each MRLC mutant. Localizations of P-MRLC in HeLa cells expressing EGFP, Wt-MRLC or each MRLC mutant at interphase (A) and cytokinesis (B). Cells were fixed with 3.7% formaldehyde and processed for confocal microscopy to detect MRLC-EGFP and P-MRLC. Bars, 50 µm (A) and 10 µm (B). Interestingly, P-MRLC is faintly detected in DD-, AD- and AA-MRLC-transfected cells. Transfected cells are marked with white asterisks in (A). (C) Schematic diagram of dividing cells are shown. The contour of entire cell region (a) and contractile ring region (b) are outlined. (D) The fluorescence intensity of P-MRLC in dividing transfected cells. The relative fluorescence intensity was defined as fluorescence intensity of P-MRLC in each MRLC mutant-expressing cell divided by that in the EGFP-expressing cell and shown in (D) (ordinate). Graph shows the relative fluorescence intensity of P-MRLC in the entire cell region (gray bars) and the contractile ring region (closed bars). Values represent standard error for at least 20 cells. (E) Western blot analysis of P-MRLC in cells expressing EGFP, Wt-MRLC or each MRLC mutant. The cell lysates of HeLa cells transfected with EGFP, Wt-MRLC or each MRLC mutant were processed for Western blot using anti-P-MRLC. Equivalent amounts of proteins were loaded on each well (see {alpha}-tubulin staining). Phosphorylated MRLC-EGFP and MRLC were detected at 46K as EGFP fused MRLCs and at 20K as intact MRLC, respectively. Similar results were obtained in two independent experiments. (F) Western blot analysis of endogenous MRLC in cells expressing each MRLC mutant. Endogenous MRLC and exogenous MRLC-EGFP were detected by A-20 at 20K and 46K, respectively. Similar results were obtained in two independent experiments.

 
Phosphorylation of MRLC is not responsible for localization of myosin II and F-actin in the contractile ring

Kosako et al. (2000) reported that Y27632, a Rho-kinase inhibitor, decreased the amount of P-MRLC localized along the cleavage furrow during cytokinesis. To further elucidate roles of P-MRLC during cytokinesis, we treated HeLa cells with Y27632. The amount of p-MRLC on the contractile ring region markedly decreased by treating with 10 µM Y27632 (Fig. 4A,B, graph a). Interestingly, the amount of F-actin localized along the contractile ring region did not change even in the presence of 100 µM Y27632 (Fig. 4A,B, graph b). The localization of P-MRLC and F-actin in the contractile ring region was further investigated in Wt- (Fig. 5A, panel a), DD- (Fig. 5A, panel b) and AA- (Fig. 5A, panel c) MRLC-expressing cells treated with or without Y27632 (Fig. 5A). In those three kinds of MRLC-expressing cells, the fluorescence intensity of P-MRLC (Fig. 5B, graph a) but not F-actin (Fig. 5B, graph b) localized along the contractile ring region decreased within 30 min after addition of 10 µM Y27632. This reveals that Y27632 suppresses the localization of P-MRLC but not F-actin along the contractile ring region in the three kinds of transfected cells. These results suggest that the localization of F-actin into contractile ring does not require P-MRLC. Next, the localization of MHC was examined in the dividing cells treated with or without Y27632. In both AA-MRLC-transfected and normal cells, MHC was observed in the contractile ring region (Fig. 6A). Quantitative analysis using fluorescence intensity of MHC revealed that the amount of MHC in the contractile ring region did not change in AA-MRLC-transfected and normal cells (Fig. 6B). To elucidate effects of Y27632 to the amount of total MRLC in the contractile ring region (Fig. 6D), time-lapse images of Wt-MRLC-EGFP-expressing cells treated with or without Y27632 during cytokinesis were obtained (Fig. 6C). As the cell division progressed, the amount of Wt-MRLC-EGFP in the contractile ring decreased both in the presence and absence of Y27632 (Fig. 6E, graphs a and b). Interestingly, both in the absence and presence of Y27632, the average amount of Wt-MRLC-EGFP from start cytokinesis to late cytokinesis in the contractile ring was the same (Fig. 6E, graph c). These results indicated that decrease of the amount of P-MRLC by Y27632 has no effect for the accumulation of myosin II and F-actin into the contractile ring.


Figure 4
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  		Figure 4  P-MRLC and F-actin localization in the contractile ring of HeLa cells in the presence of various concentrations of Y27632, a Rho-kinase inhibitor. (A) Immunofluorescence staining of P-MRLC and F-actin in nontransfected cells treated with each concentration of Y27632, and processed for confocal microscopy. Bar, 10 µm. (B) Quantitative analysis of the concentration of P-MRLC (a) and F-actin (b) in the contractile ring region (as shown in Fig. 2B panel b) of HeLa cells in the presence of each concentration of Y27632. Each horizontal bar is shown as an average of at least 12 cells in each concentration of Y27632.

 

Figure 5
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  		Figure 5  Effects of Y27632 on the localization of P-MRLC and F-actin in the contractile ring of MRLC mutant-expressing cells. (A) Nonsynchronized HeLa cells expressing Wt-MRLC (a), DD-MRLC (b) or AA-MRLC (c) were treated with or without 10 µM Y27632 for indicated time, fixed with 3.7% formaldehyde, and observed using conventional microscope to detect P-MRLC and F-actin. Observations were made on the cells that happened to be undergoing cytokinesis when fixed after appropriate incubation time following the addition of the Y27632. Bar, 10 µm. (B) Graphs show fluorescence intensity of P-MRLC (a) and F-actin (b) on the contractile ring region of HeLa cells in each time after addition of 10 µM Y27632. Values represent standard error for at least 7 cells. Fluorescence intensity of P-MRLC and F-actin in the contractile ring region of each dividing cell (boxed with dotted line in (A, panel a)) were detected and shown in (B) (ordinate). The fluorescent intensities indicated on time 0 are the control which are added just DDW as the solvent of Y27632.

 

Figure 6
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  		Figure 6  Localization of MHC and exogenous Wt-MRLC on the contractile ring in dividing cells treated with or without 10 µM Y27632. (A) AA-MRLC-expressing and nontransfected cells were treated with or without Y27632 for 30 min, fixed with 3.7% formaldehyde, stained for MHC and observed using confocal microscope. Bar, 10 µm. (B) Graph shows fluorescence intensity of MHC in the contractile ring region (as shown in Fig. 2B, panel b) of HeLa cells treated with or without Y27632 for 30 min. Values represent standard error for at least 20 cells. (C) Fluorescence images by conventional microscope of each progress of furrow ingression in mitotic HeLa cells over-expressing EGFP-tagged Wt-MRLC treated with or without Y27632. The width of cleavage furrow defined as a relative diameter (as shown in Fig. 1B) is shown in each panel. Bar, 10 µm. (D) Schematic diagram of dividing cells are shown. The contour of the entire cell region (a) and the contractile ring region (b) are outlined. The percentage of fluorescence intensity in (b) divided by that in (a) was represented as MRLC-EGFP accumulation ratio in (E) (ordinate). (E) The amount of MRLC-EGFP on the contractile ring region in Wt-MRLC-expressing cells treated with Y27632 during cytokinesis. Graphs show MRLC-EGFP accumulation ratio in the dividing cells treated with (b) or without (a) Y27632. Abscissa shows the width of cleavage furrow as relative diameter (as shown in Fig. 1B). Each symbol represents an individual cell (observed nine cells for (a) and three cells for (b) obtained from three independent experiments). Graph (c) shows the mean values of accumulation ratio of each cell transfected with Wt-MRLC-EGFP in the absence (a) or presence (b) of Y27632 from start cytokinesis to late cytokinesis.

 
Phosphorylation of MRLC plays important roles to control the progress of furrow ingression

To further elucidate how P-MRLC in the contractile ring controls furrow ingression, its progress in each MRLC-transfected cell treated with Y27632 was analyzed. Figure 7A shows the progress of furrow ingression in each MRLC-transfected cell treated with or without 10 µM Y27632. The furrow ingression of Wt- and AA-MRLC-expressing cells treated with Y27632 was slower than that in the absence of Y27632 (Fig. 7A, graphs a and d). By contrast, in DD- and AD-MRLC-expressing cells, Y27632 showed no effect on the furrow ingression (Fig. 7A, graphs b and c). In Fig. 7B, the time from start cytokinesis to 50% cytokinesis was investigated in dividing Wt- and AD-MRLC-expressing cells treated with various concentrations of Y27632. As shown in Fig. 7B, AD-MRLC (graph b) but not Wt-MRLC (graph a) cancelled the effect of Y27632 to suppress the furrow ingression. These results suggest that phosphorylation of MRLC is important to control the speed of furrow ingression in dividing HeLa cells.


Figure 7
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  		Figure 7  The progress of furrow ingression of MRLC mutant-expressing cells treated with Y27632. (A) HeLa cells expressing Wt-MRLC or each MRLC mutant were treated with or without 10 µM Y27632. Graphs show the progression of cell division of HeLa cells expressing Wt-MRLC (a), DD-MRLC (b), AD-MRLC (c) or AA-MRLC (d). Ordinate shows the width of cleavage furrow as relative diameter (as shown in Fig. 1B). Each control (without Y27632) shown with solid line is plotted as average of at least 10 cells. The result in cells treated with Y27632 (dotted line) is a representative in at least three independent experiments. (B) The graphs show the time from start cytokinesis to 50% cytokinesis in dividing Wt-MRLC- (a) and AD-MRLC- (b) expressing HeLa cells treated with each concentration of Y27632. Error bars represent mean values in at least three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Here, EGFP-tagged wild-type and phospho-mimic MRLCs were expressed in HeLa cells (Uchimura et al. 2002). All of HeLa cells expressing four kinds of recombinant MRLCs observed in this experiments (n = 30 cells for each) did not show cytokinesis failure, such as regression or no-furrowing (data not shown). Previous experiments have indicated that the cells expressing AA-MRLC caused multinucleation (Komatsu et al. 2000; Dean & Spudich 2006). In Drosophila cells, however, less than 25% of cells expressing AA-MRLC showed multinucleation both in the presence and absence of endogenous MRLC (Dean & Spudich 2006). In other words, more than 75% of cells expressing AA-MRLC successfully divided. This supports our present data. In our studies, the cells expressing AA-MRLC showed a delay of furrow ingression of dividing cells. This is the first direct demonstration that phosphorylation of MRLC is important to control the speed of furrow ingression in mammalian cultured cells.

Live-cell imaging revealed that nonphosphorylatable form of MRLC, as well as the phospho-mimic MRLC, was localized to the contractile ring in dividing cells (Miyauchi et al. 2006 and the present study). Dean & Spudich (2006) reported that the cells expressing AA-MRLC does not show the accumulation of the AA-MRLC into the equatorial cortex of no furrowing cells. Although no images showing the localization of AA-MRLC in furrowed cells are available in the report, AA-MRLC might localize at the furrow in cells that successfully divided, as shown in this manuscript. Previous experiments using EGFP- and myc-tagged MRLCs revealed that nonphosphorylatable MRLC was dispersed from the furrow in 50%–90% furrowed cells, although phospho-mimic MRLC was retained in the furrow (Iwasaki et al. 2001; Miyauchi et al. 2006). In this manuscript, living cell-imaging also revealed that phospho-mimic MRLC was retained in the furrow, although AA-MRLC was dispersed from the furrow (Fig. 1A). This supports our previous studies indicating that phosphorylation of MRLC is required for retainment but not recruitment of myosin II on the furrow during furrow ingression.

Here we showed that the localization of MHC and F-actin along the furrow was not dependent on the phosphorylation state of MRLC because these localizations were not affected by the expression of AA-MRLC (Figs 1, 3 and 6). This simply suggests that the phosphorylation of MRLC is not important to retain myosin II and F-actin in the furrow. However, the amount of AA-MRLC in the furrow was diminished (Fig. 1A), allowing an alternative interpretation that the endogenous MRLC in the furrow was sufficient to retain MHC and F-actin in the furrow. Even so, the expression of four kinds of MRLC-EGFP decreased the amount of P-MRLC (Fig. 3E), suggesting that the endogenous MRLC that retained MHC and F-actin in the furrow was nonphosphorylated. This view is supported by the observation that MHC and F-actin were normally retained in the furrow even in the presence of Y27632, which efficiently inhibited phosphorylation of endogenous MRLC.

In higher eukaryotic cells, phosphorylation of MRLC seems to be generally required for cytokinesis. However, MRLC kinases used preferentially are different among cell types, although at present we do not know how such difference among cell types arises. Myosin light chain kinase (MLCK), rather than Rho kinase, is required for cytokinesis in sea urchin eggs (Uehara et al. 2008) and Rat1 cell (Yoshizaki et al. 2004). On the other hand, Rho kinase, rather than MLCK, seems to play a predominant role in cytokinesis in HeLa cell (Kosako et al. 2000; Yoshizaki et al. 2004) and Drosophila S2 cell (Dean et al. 2005; Dean & Spudich 2006; Hickson et al. 2006). In our experiments, HeLa cells treated with Y27632 showed a significant delay of furrow ingression. As shown in Fig. 4A, phoshporylation of MRLC at the cleavage furrow was strongly inhibited by Y27632. However, expression of phospho-mimic but not nonphosphorylatable MRLC completely rescued the defect for furrow ingression in Y27632 treated cells (Fig. 7), suggesting that the major role of Rho kinase during cytokinesis is to control the amount of P-MRLC by phosphorylation of MRLC or myosin phosphatase in the cell. Further, it has been elucidated that Rho-mediated signaling is required for localization of P-MRLC in the contractile ring (Kamijo et al. 2006). These results suggest that the MRLC phosphorylated by Rho-mediated signaling plays important roles to control the speed of furrow ingression during cytokinesis in HeLa cells.

How is P-MRLC retained in the furrow during furrow ingression? Accumulating evidence obtained from many cells suggests that myosin II localizes to the furrow independently of F-actin (Schroeder & Otto 1988; Zang & Spudich 1998; Naqvi et al. 1999; Motegi et al. 2004). Recent studies revealed that SEPT2, one of mammalian septins, and the contractile ring protein anillin interact directly with myosin II and that these interaction is regulated by MRLC phosphorylation (Straight et al. 2005; Joo et al. 2007). When SEPT2 was dissociated from myosin II, a significant decrease in the P-MRLC and dissociation of the myosin II from myosin II kinases, such as Rho-kinase or Citron-kinase, were observed. Further, phosphorylation of MRLC is required for anillin binding to myosin II. These results suggest that SEPT2 (containing septin filaments) and/or anillin act as a scaffold for myosin II and its kinase to ensure the activation of myosin II at the furrow. Further studies will be needed to show whether these scaffold proteins are essential for a proper spatiotemporal regulation of MRLC phosphorylation during furrow ingression in cytokinesis.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Antibodies and reagents

The rabbit anti-MHC polyclonal antibody was kindly provided by Dr K. Fujiwara (University of Rochester, Rochester, NY). Anti-chick brain {alpha}-tubulin monoclonal antibody (DM1A), anti-P-MRLC polyclonal antibody (#3671) and anti-MRLC polyclonal antibody (A-20) were purchased from Seikagaku Corporation (Japan), Cell Signaling Technology (USA) and Santa Cruz Biotechnology (USA), respectively. F-actin was localized TRITC-phalloidin, FITC-phalloidin or CPITC-phalloidin (Sigma Chemical, USA). Alexa Fluor 568 goat anti-rabbit IgG (H + L) antibody was purchased from Molecular Probes (USA). Alkaline phosphatase conjugated anti-mouse IgG (H + L) antibody and Alkaline phosphatase conjugated anti-rabbit (Fc) IgG antibody were from Promega (USA). Alkaline phosphatase conjugated anti-goat IgG (H + L) antibody was from Chemicon (USA). Y27632 was from Welfide Corp (Japan).

Cell culture

HeLa cells (RCB0007) were obtained from the Riken Cell Bank (Japan). They were cultured in Eagle's Minimal Essential Medium (MEM; Nissui, Japan) containing 10% fetal bovine serum (FBS; Intergen, USA).

Plasmids and transfection

The wild-type MRLC cDNA and MRLC mutants (termed as DD-, AD- and AA-MRLC) cDNAs were subcloned into the pEGFP-N1 vector (Clontech, USA) (Uchimura et al. 2002). For transfection, HeLa cells were incubated at 37 °C for 4 h in 1 mL of OPTI-MEM containing DNA with 6 µL of LipofectAMINE (Gibco BRL, USA). Then the medium was replaced with MEM containing 10% FBS and the cells were incubated for 24 h. Cells were treated as described above.

Immunofluorescence

Cells on coverslips were fixed with 3.7% formaldehyde in PBS (140 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4 and 8 mM Na2HPO4 (pH 7.3)) for 15 min, and permeabilized with 0.2% Triton X-100 in PBS for 4 min at room temperature. After washing four times with PBS, the coverslips were incubated with the rabbit anti-P-MRLC polyclonal antibody (1 : 20) or the rabbit anti-MHC polyclonal antibody (1 : 150) for 1 h at 37 °C. Cells were washed four times with PBS and then incubated with Alexa Fluor 568 goat anti-rabbit IgG (H + L) antibody (1 : 200) for 1 h at 37 °C. For observing actin filaments, cells were stained with TRITC-phalloidin (1 : 200), FITC-phalloidin (1 : 200) or CPITC-phalloidin (1 : 100) for 1 h at 37 °C. Then cells were washed four times with PBS and mounted on to a microscope slide with a drop of Fluoro GuardTM. (Bio-Rad, USA) for preserving fluorescence. Specimens were observed using a Nikon ECLIPSE TE300 microscope. Images were recorded with a digital CCD camera (ORCA, C4742-95-12; Hamamatsu Photonics, Japan). Specimens were also analyzed using a confocal microscope LSM410 (Carl Zeiss, Germany). For quantitative analyses, acquired images were analyzed on NIHimage 1.63 software (National Institutes of Health, USA).

Analysis of living cells

For living cell analysis, HeLa cells were cultured on glass-based dishes (IWAKI, Japan) for 24 h. Then transfection was performed as described above. After additional incubation for 24 h, these cells were observed using a Nikon ECLIPSE TE300 microscope equipped with the moist chamber at 37 °C.

Electrophoresis and Western blot

Transfected cells were homogenized with 2x SDS sample buffer (200 mM Tris-HCl (pH 6.8), 250 mM dithiothreitol, 5% SDS and 22.5% glycerol with an adequate amount of bromophenol blue). Each sample was separated by SDS-PAGE, which was preformed according to the method of Blattler et al. (1972) with 12% polyacrylamide slab gels, except that the buffer system of Laemmli (1970) was used. Then the proteins were transferred on to PVDF membrane (pore size 0.2 µm) (Millipore, USA) using a transfer buffer (40 mM glycine, 48 mM Tris base). Membranes were blocked with 1% BSA or 5% skim milk dissolved in PBS and incubated with primary antibody against {alpha}-tubulin (1 : 500), MRLC (1 : 100) or P-MRLC (1 : 100) for 1 h at 37 °C. After washing four times with PBS containing 0.02% Tween-20, membranes were incubated with secondary antibody conjugated with alkaline phosphatase (1 : 5000). The antigen was detected by amplifying with Nitro Blue tetrazolium and BCIP (Nacalai Tesque, Japan).

Drug treatment

Y27632 was dissolved in DDW and stocked at –30 °C until use. Cells were incubated with Y27632 at the final concentration of 10–100 µM for 5 or 30 min prior to live imaging or fixation.


   Footnotes
 
Communicated by: Shuh Narumiya

* Correspondence: hhosoya{at}hiroshima-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 1 January 2009
Accepted: 12 February 2009





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