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Vol. 17, Issue 9, 4093-4104, September 2006
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*Department of Biology, New Mexico State University, Las Cruces, NM 88003; Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; and Marine Biological Laboratory, Woods Hole, MA 02543
Submitted February 9, 2006;
Revised June 15, 2006;
Accepted July 5, 2006
Monitoring Editor: Yu-li Wang
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Force generation is accomplished by the transient assembly of a contractile ring, and recent genetic and proteomic approaches have significantly increased our knowledge of ring components and regulatory molecules (Echard et al., 2004; Eggert et al., 2004; Skop et al., 2004). Studies in yeast using green fluorescent protein-tagged ring components have demonstrated that ring components are recruited to the division site in a hierarchical manner that involves the progressive assembly of more complex structures (Lippincott and Li, 1998; Wu et al., 2003; Wu and Pollard, 2005). In contrast, the temporal sequence in which ring components are recruited to the division site in animal cells is not as well resolved; thus, it remains unclear whether the contractile ring assembles in a similar sequential manner. Moreover, it is not known how ring assembly is coordinated in space and time with chromosome segregation. Thus, although great advances have been made in our understanding of the contractile ring itself, fundamental gaps remain in our understanding of the spatiotemporal regulation of cytokinesis.
The mechanical nature of cytokinesis was appreciated by early cell biologists (Wilson, 1928; Rappaport, 1996), and for decades investigators have taken advantage of the large size and regular geometry of echinoderm eggs to study the biophysical changes in the cell surface during cell division. Using a variety of quantitative methods, changes in cortical tension were observed leading up to and during cytokinesis, but their timing relative to the nuclear division cycle or the physiological significance of these changes was unknown (Mitchison and Swann, 1955; Hiramoto, 1963, 1970, 1990; Yoneda and Dan, 1972; Yoneda et al., 1978; Yoneda and Schroeder, 1984; Yoneda, 1986). A common observation among many of these studies is a sharp, transient increase in cortical tension several minutes before furrow initiation (Yoneda and Dan, 1972; Usui and Yoneda, 1982; Ohtsubo and Hiramoto, 1985), which has been attributed to an increase in cortical actin (Usui and Yoneda, 1982). Simultaneous monitoring of polar and equatorial surfaces in sea urchin eggs (Ohtsubo and Hiramoto, 1985), and later in cultured mammalian cells, (Burton and Taylor, 1997; Matzke et al., 2001) revealed a dramatic but regionally restricted increase in tension at the cell equator. Although these physical changes were probably produced due to a balance between equatorial contractility and polar cortical tension (Robinson and Spudich, 2000; Girard et al., 2004; Reichl et al., 2005; Wang, 2005), the primary mediator of these physical changes is myosin II.
Myosin II is the force-generating motor for cytokinesis and is subject to regulation by both heavy and regulatory light chain phosphorylation (Matsumura et al., 2001; Matsumura, 2005). Our understanding of heavy chain phosphorylation comes primarily from studies on the slime mold Dictyostelium discoideum, where phosphorylation of the tail region by a family of heavy chain kinases inhibits filament formation (Egelhoff et al., 1993; Sabry et al., 1997; Yumura and Uyeda, 1997; Zang and Spudich, 1998; Yumura et al., 2005). The expression of nonphosphorylatable heavy chain mutants results in abnormally high myosin II recruitment to the cell equator, whereas expression of myosin II heavy chains containing amino acid substitutions that mimic the phosphorylated state prevents recruitment to the contractile ring (Sabry et al., 1997; Yumura, 2001). These phosphorylation sites reside in a C-terminal extension of myosin heavy chain that is required for proper cytokinesis in Dictyostelium (O’Halloran and Spudich, 1990) but is absent from myosin II in animal cells. In mammalian cells, protein kinase C and casein kinase II phosphorylate myosin II heavy chain (Murakami et al., 1998, 2000; Bresnick, 1999; Dulyaninova et al., 2005), and the S100 family protein metastasin 1 binds to and prevents myosin IIA filament assembly (Li et al., 2003). However, the functional significance of the regulatory mechanisms during cell division remains unclear.
Phosphorylation of the myosin regulatory light chain (MRLC) both positively and negatively regulates myosin II, and both mechanisms have been implicated in regulating myosin II during cytokinesis (Satterwhite et al., 1992; Bresnick, 1999; Matsumura, 2005). Light chain phosphorylation on Ser19 and Thr18 activates myosin by promoting both myosin/actin interactions and filament assembly (Scholey et al., 1980; Ikebe et al., 1988) and nonphosphorylatable mutants of MRLC block cytokinesis in Drosophila and mammalian cells (Jordan and Karess, 1997; Simerly et al., 1998; Komatsu et al., 2000). Multiple kinases have been implicated in mediating MRLC phosphorylation on these sites, either by directly phosphorylating Ser19 or by inhibiting myosin phosphatase (Matsumura, 2005). Although the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Rho-kinase (ROCK) are likely candidates, genetic or chemical inhibition studies have implicated only citron kinase, and it seems to function late in cytokinesis (Echard et al., 2004; Naim et al., 2004). Therefore, the kinase or kinases mediating myosin light chain phosphorylation early in cytokinesis have not been fully elucidated.
To examine the timing of myosin II contractility in living cells as well as dissect the signals that influence myosin II activation, we have developed a compression assay that detects subtle changes in the isotropic cytoskeleton of sea urchin eggs (Stack et al., 2006). We report here that myosin II is transiently and globally activated before cleavage plane specification. Moreover, myosin II activation during cell division requires multiple inputs from different signaling pathways, giving rise to a model whereby calcium-dependent signaling mediates the global activation of myosin, and Rho-dependent signaling focuses on contractility for cytokinesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Briefly, at 10 min after fertilization, eggs were washed with hypertonic CaFSW (8 ml of 2.5 M NaCl added to 50 ml CaFSW, pH 8.9) for 15 min and then washed back with isotonic CaFSW.
Chemicals and Reagents
Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Blebbistatin, nocodazole, ML-7, and H-1152 were purchased from Calbiochem (San Diego, CA). Rho-guanine nucleotide dissociation inhibitor (Rho-GDI) was purchased from Cytoskeleton (Denver, CO), and P4(5)-(1-(2-nitrophenyl)-ethyl) ester (NPE)-caged inositol-(1,4,5)-trisphosphate (cIP3) and calcium-green dextran (mol. wt. 10,000) were purchased from Invitrogen (Carlsbad, CA). The antennapedia-MLCK inhibitory peptide (RQIKIWFQNRRMKWKK AKKLSKDRMKKYMARRKWQKTG) was synthesized at the Tufts University Core Facility (Boston, MA).
Micromanipulation
To observe changes in cortical contractility during the cell cycle, eggs were placed under compression using a gas-permeable fluorocarbon oil (Sluder et al., 1999; Stack et al., 2006). Briefly, fertilized eggs stripped of their fertilization envelopes were placed in a glass-bottomed 35-mm culture dish (WPI, Sarasota, FL) precoated with 10 mg/ml protamine sulfate (Sigma-Aldrich) and allowed to settle for 5 min. The eggs were compressed 40 min after fertilization by rapidly removing seawater and replacing with 1.5 ml of Fluorinert FC-40 oil (Sigma-Aldrich). Measurements of cell geometry were performed using ImageJ (http://rsb.info.nih.gov/ij/).
Microinjection
All reagents for microinjection were resuspended in injection buffer (10 mM HEPES and 150 mM sodium aspartate, pH 7.0) and back-filled into glass capillary pipettes (WPI). In all injection experiments, cells were injected with 
10–40 pl (1.4–5.7% of cell volume) before withdrawal of seawater and replacement of FC-40 oil.
To visualize chromosome segregation in living sea urchin embryos, eggs were microinjected at the single-cell stage shortly after fertilization with 1 mg/ml calf histone labeled with Alexa Fluor-488 (Protein Labeling kit; Invitrogen). For calcium mobilization experiments, eggs were microinjected with 2 mM calcium green dextran and 2 mM NPE-cIP3 under safelight conditions 10–15 min after fertilization. Injected eggs were then compressed under Fluorinert FC-40 oil 10 min after microinjection and observed by differential interference contrast (DIC)/ fluorescein isothiocyanate (FITC) fluorescence at 10-s intervals through nuclear envelope breakdown, after which cells received a single 500-ms pulse of UV light to uncage the IP3 and mobilize intracellular calcium.
Detection of Ser19 Phosphorylation In Vivo
Lytechinus pictus eggs were fertilized, stripped of their fertilization envelopes and cultured up until the first mitosis. Cells were then fixed and permeablized using a protocol optimized for visualizing the actin cytoskeleton (Wong et al., 1997). Cells were then washed, blocked with 3% bovine serum albumin in phosphate-buffered saline/2 mM NaF, and processed for tubulin (DM1A; Sigma-Aldrich), activated myosin II [rabbit or mouse anti-phospho(Ser19)-myosin regulatory light chain; Cell Signaling Technology, Beverly, MA], or egg myosin II localization. Primary antibodies were detected with Alexa Fluor-labeled secondary antibodies (Invitrogen), and confocal images were acquired using an Olympus FluoView confocal microscope (Olympus America, Melville, NY) at the Central Microscopy Facility at the Marine Biological Laboratory (Woods Hole, MA).
Imaging Acquisition and Processing
All live cell experiments were performed on Zeiss Axiovert 200M microscopes equipped with computer-driven Uniblitz (Vincent Associates, Toronto, Ontario, Canada) and internal fluorescence shutters to control bright field and epifluorescence light sources. Brook Industries (Lake Villa, IL) heating/cooling stage inserts were used to maintain sample temperature at 15°C. DIC and fluorescence images were recorded using a 12-bit Axiocam charge-coupled device (CCD) camera driven by AxioVision 4.2, and figures were prepared using ImageJ version 1.34, Adobe Photoshop 6.0.1 (Adobe Systems, Mountain View, CA), and QuickTime software (Apple Computer, Cuptertino, CA). For polarization microscopy, an Axiovert 200M was configured with a circular polarizer and 546-nm filter placed above the condenser, and a liquid crystal universal compensator (LC-Polscope; Cambridge Research Instruments, Woburn, MA) was placed below the reflector turret (Oldenbourg and Mei, 1995). An EXFO X-Cite 120 light source (Exfo Life Sciences, Mississauga, Ontario, Canada) was used for transillumination, and images were acquired using a Q Imaging cooled CCD camera controlled by PSJ software (Marine Biological Laboratory). Image stacks were acquired using a 3-nm retardance ceiling, exported as 8-bit tiffs to ImageJ, where kymographs as well as three-dimensional surface projections were generated. Figures were prepared with Adobe Photoshop 6.0.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Rappaport, 1996). Other studies in sea urchin eggs suggested that the acquisition of furrow-inducing capacity or cortical responsiveness occurs abruptly at the metaphase–anaphase transition (Shuster and Burgess, 2002b), and we sought to develop a physical manipulation that would detect early events leading up to cytokinesis. A classical method for studying changes in cortical contractility in echinoderm eggs has been to measure resistance to compression (Cole, 1932; Cole and Michaelis, 1932; Yoneda, 1964; Yoneda, 1986). More recently, we found that a common method for observing mitosis in sea urchin eggs (Sluder et al., 1999) provided a sensitive and convenient microscopic assay for detecting myosin II-dependent, blebbistatin-sensitive changes in cortical contractility during fertilization (Stack et al., 2006). As shown in Figure 1, sea urchin blastomeres plated onto glass-bottomed culture dishes could be flattened from a diameter of 110 µm to a thickness of 18–30 µm by rapidly exchanging seawater with a gas-permeable fluorocarbon oil. Cells flattened in this manner remained viable for up to 8 h and continued to divide, although extremely flat cells frequently failed to complete cytokinesis. Cells manipulated in this manner were followed through the first cell cycle in the absence (Figure 1, A–D, top) or presence of 50 µM blebbistatin (Figure 1, E–H, top), an inhibitor of nonmuscle myosin II ATPase activity (Straight et al., 2003; Kovacs et al., 2004) that also blocks myosin contractility in cytokinesis in sea urchin eggs (Ng et al., 2005; Stack et al., 2006). As shown in Figure 1, A–D (and Supplemental Videos 1 and 6), in control cells there was a sharp increase in cortical tension 13 min after nuclear envelope breakdown (NEB), as evidenced by the decrease in cell diameter as the cell resisted compression (Figure 1B). The initial increase in cortical contractility was followed by a brief relaxation before the onset of furrowing (Figure 1D). The amplitude of this prefurrow "contraction" was roughly proportional to the degree of flattening, with less pronounced contractions seen in cells flattened to a thickness of 
30 or <17 µm. However, in each case (n = 45), a cortical contraction preceded the onset of furrowing. In contrast, blebbistatin suppressed both the initial increase in contractility, and the cleavage furrow in a dose-dependent manner (Figure 1, bottom), suggesting that the increased resistance to compression was dependent on myosin II (Figure 1, E–H, and Supplemental Video 2). Although off-target effects have been reported (Shu et al., 2005), blebbistatin was effective at concentrations between 25 and 100 µM (Figure 1, bottom) without affecting F-actin assembly and organization, cortical granule release, or calcium signaling (Stack et al., 2006).
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). To better visualize chromosome segregation in cells under compression, fertilized eggs were injected with fluorescently labeled histone before flattening and nuclear envelope breakdown, and followed through the first and second cell divisions (Figure 2 and Supplemental Video 3). Simultaneous imaging of chromosome segregation and contractility revealed that in each case (n = 9), contractility initiated 60 s before the first visible poleward chromatid movements (Figure 2E, bracket). Thus, the ability to unambiguously follow cortical contractility relative to the nuclear clock allowed us to assign the onset of myosin II contractility to the metaphase–anaphase transition.
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). To examine myosin activation in vivo, fertilized eggs were fixed and processed for microtubule and phospho-Ser19 MRLC localization (Figure 3) by using an antibody previously demonstrated to react with phosphorylated sea urchin myosin regulatory light chain (Stack et al., 2006). Roughly half (30/55 cells scored) of the cells in metaphase (75 min after fixation) had a bright cortical rim of Ser19 phosphorylation (Figure 3, A–F). This contrasted sharply with cells in anaphase or cytokinesis, where light chain phosphorylation was confined to the cleavage plane (Figure 3, G–L). Although the level of cell synchrony was not sufficient to resolve early versus late metaphase (Figure 3, A–C), the presence of activating myosin II phosphorylation in the cortex of cells with aligned chromosomes (our unpublished data) and a well-developed mitotic apparatus supported our findings in living cells that there was an increase in contractility at the metaphase–anaphase transition (Figures 1 and 2).
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; Burgess and Chang, 2005), and there is now general agreement that microtubules are required for cleavage plane determination as well as the maintenance of the cleavage furrow and abscission (Wheatley and Wang, 1996; Larkin and Danilchik, 1999). Although the molecular nature of the cleavage stimulus remains unknown, recent studies in sea urchin eggs demonstrated a microtubule-dependent localization of active Rho GTPase at the cleavage plane before furrow initiation (Bement et al., 2005). The increase of cortical contractility at anaphase onset (Figures 1 and 2) seemed to precede cleavage plane determination, which normally occurs 1 min after contact between the cortex and the astral arrays (Rappaport and Ebstein, 1965) or 7 min after anaphase onset in L. pictus eggs (Shuster and Burgess, 2002b). To examine the timing of myosin II activation relative to cleavage plane determination, spherical and flattened eggs were followed using liquid crystal polarization microscopy (Oldenbourg and Mei, 1995). In the unflattened cell shown in Figure 5, A–D, a sharp drop in spindle birefringence was detected within 30 s after anaphase onset, as astral microtubules elongate and contact the cell cortex. Approximately 7 min after anaphase onset, the first detectable surface activity was observed at the cell equator (Figure 5, C and D, Supplemental Video 4). When flattened cells were examined (Figure 6 and Supplemental Video 5), ingression of the cell margin preceded the transition in microtubule dynamics by 60 s (Figure 6E). This was visible both in the raw image as well as in a surface plot of retardance values (Figure 6, insets, and Supplemental Video 5), where the ingression of the cell margins clearly preceded the drop in spindle birefringence. Although the increase in cortical contractility was closely tied to anaphase onset, it appeared that the increase in myosin II contractility preceded the stabilization of microtubule dynamics and cleavage plane determination.
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) and that the localization of the contractile ring arises from a local deficit of microtubules at the equatorial plane (Schroeder, 1972; Dechant and Glotzer, 2003). To further explore any possible role that microtubules might play in regulating cortical contractility, cells were treated before metaphase with reagents that modulate microtubule dynamics or density (Figure 7). To reduce microtubule polymer in cells, cells were treated with 50 µM nocodazole (Figure 7B) to prevent spindle assembly. Although reduction or depolymerization of the mitotic apparatus can delay anaphase and mitotic exit in sea urchin eggs (Sluder, 1976, 1979), nocodazole did not significantly suppress the increase in myosin II contractility (Figures 1, bottom, and 7B', and Supplemental Video 7); however, the subsequent relaxation observed in control cells did not occur in nocodazole treated cells (n = 10) (compare Figures 7A' and 6B', respectively). To examine whether astral microtubule–cortex interactions could suppress prefurrow contractility, fertilized eggs were treated briefly with hypertonic seawater, which results in de novo centriole formation (Kuriyama and Borisy, 1983; Kallenbach, 1985; Sluder et al., 1997). As shown in Figure 7, C and C' (and Supplemental Video 8), as many as 35 supernumery asters (or cytasters) appeared at the onset of mitosis. The presence of microtubule asters throughout the cytoplasm failed to suppress prefurrow contractility; however, the amplitude of the contractile response was slightly dampened relative to controls (Figures 1, bottom, and 7A, and Supplemental Movie 6). Thus, the activation of prefurrow cortical contractility appeared to be independent of microtubules, but the transient relaxation before furrow formation was sensitive to microtubule depletion.
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–5). These data fit earlier predictions suggesting that the proximal signals for cytokinesis are triggered at the metaphase–anaphase transition (Shuster and Burgess, 1999, 2002b). As mentioned, MLCK, ROCK, and citron kinases have been implicated in the regulation of myosin II during cytokinesis (Matsumura et al., 2001, 2005), but citron kinase has been shown to act primarily at the end of cytokinesis. To identify the signaling pathways responsible for myosin II activation at the metaphase–anaphase transition, cells were treated or microinjected with reagents that inhibit either the Rho- or Ca2+/MLCK signaling pathways (Figures 1, bottom, and 8). Microinjection of Rho-GDI, which binds GDP-Rho and inhibits exchange of GDP for GTP, resulted in a nearly complete loss of cortical tension (n = 7), with cells unable to resist compression (Figure 8, A and A', and Supplemental Video 9). Similar results were observed with C3 transferase another Rho inhibitor (our unpublished data). However, a small, but detectable increase in contractile activity was detected at anaphase onset (Figure 8, A and A'). In cells treated with the ROCK inhibitor H-1152, the timing of nuclear envelope breakdown, metaphase–anaphase transition and prefurrow contractility was not significantly affected (Figure 1, bottom), although furrowing was inhibited (n = 26) (Figure 8, B and B', and Supplemental Video 10).
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) or were treated with ML-7 (Figure 8, D and D', and Supplemental Movie 12). Complete inhibition of prefurrow contractility was observed with both the peptide and small molecule inhibitors (n = 8 and 7, respectively) (Figures 1, bottom, and 8, C' and D'). These results suggested that the initial increase in cortical contractility was a MLCK-dependent but not ROCK-dependent process.
Rho GTPase is thought to direct contractile ring assembly, via the regulation of formins and myosin II contractility (Piekny et al., 2005). However, our data indicate that ROCK was not required for prefurrow myosin II activity (Figure 8). Additionally, the role of MLCK during cytokinesis as well as the relevance of global contractile events remains unclear. Therefore, to confirm that these signaling pathways are essential for cytokinesis in sea urchin eggs, unflattened cells were treated with H-1152 or ML-7 and subsequently scored for cleavage (n = 33 for each condition). Both ROCK- and MLCK-inhibited cells failed to complete cytokinesis as indicated by the binucleated cells in Figure 9, suggesting that both ROCK and MLCK signaling are necessary for proper cytokinesis in this cell type.
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; Miller et al., 1993; Chang and Meng, 1995; Muto et al., 1996; Webb et al., 1997; Saul et al., 2004; Wong et al., 2005), and myosin activation (Shuster and Burgess, 2002a; Stack et al., 2006) as well as membrane addition (Shuster and Burgess, 2002a) are candidate downstream effectors. Imaging of calcium dynamics in dividing L. pictus eggs revealed that there is a mobilization of calcium at the metaphase–anaphase transition that is necessary for cytokinesis (Groigno and Whitaker, 1998), raising the possibility that this calcium transient may partially serve as the "timer" for the activation of myosin II. Thus, to establish whether calcium acts as an early signal for myosin II activation, fertilized eggs were injected with calcium green dextran and cIP3 before flattening. cIP3 was uncaged with a single pulse of UV light 3 min after NEB, when the cell was in early metaphase, and the calcium transient were detected by an increase in calcium green fluorescence. Uncaging of cIP3 resulted in a detectable contractile response within one minute of the UV light pulse (Figure 10, bracket, and Supplemental Video 13) (n = 6), whereas exposure to UV light in the absence of cIP3 induced no contractile response (Stack et al., 2006). Thus, even though the cell was in metaphase, at a time when myosin contractility is thought to be suppressed (Satterwhite et al., 1992), precocious mobilization of calcium was sufficient to induce a cortical contractile response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wu et al., 2003; Wu and Pollard, 2005). Although the late events of cytokinesis seem to be well resolved, the sequence of early events leading to ring assembly in animal cells remains unclear. In an effort to address these issues, a simple yet sensitive manipulation of living cells was used to resolve the temporal and spatial dynamics of myosin II activation. The global activation of myosin II detected at the metaphase–anaphase transition is consistent with both the early biophysical studies of cytokinesis (Hiramoto, 1990; Rappaport, 1996) as well as more recent studies defining the earliest window of furrowing capacity (Rappaport and Rappaport, 1993; Shuster and Burgess, 2002b). Moreover, a recent study that measured forces at the surface of dividing sea urchin eggs reported a sharp increase in cortical tension 6 min before furrow initiation (Miyoshi et al., 2006). This initial increase in tension was isotropic and transient, with cortical tension rising again just before the onset of furrowing and oriented in the plane of the contractile ring. Force production by these "prefurrow spikes" ranged between 1.5 and 3 nN/µm for the three species tested (Clypeaster japonicus, Hemicentrotus pulchrerrimus, and Scaphechinus mirabilis). Although the compression method described here does not quantify changes in cortical tension, it allows for a more accurate measurement of the temporal coordination between chromosome segregation and prefurrow contractility (Figure 2). Lastly, these two independent methods (needle deflection and compression) demonstrate that cortical contractile activity is dependent on myosin II and MLCK. Thus, these studies shed novel insight not only into the molecular mechanisms contributing to myosin II activation but also the order of events leading up to the establishment and ingression of the cleavage furrow.
Myosin II Activation Precedes Cleavage Plane Determination
Recent studies in sea urchin eggs demonstrated that before furrow ingression, a zone of active Rho GTPase (Bement et al., 2005) as well as a lipid raft markers (Ng et al., 2005) accumulate at the cell equator in a microtubule-dependent manner, but otherwise the sequence of early events leading to contractile ring formation is poorly resolved in animal cells. In sea urchin eggs, anaphase onset is accompanied by a dramatic elongation of microtubules toward the cortex, and active furrowing can be detected after a short latent period (Rappaport and Ebstein, 1965; Strickland et al., 2005). Although species and temperature dependent, the total time from anaphase onset to the initiation of furrowing in L. pictus has been measured to be 7.5 min (Shuster and Burgess, 2002b). Mapping of contractile activity by compression revealed that myosin II activation coincides with the metaphase–anaphase transition (Figure 2), which considerably precedes the outgrowth of astral microtubules (Figure 6). Localization studies of microtubules and Ser19-phosphorylated light chain (Figure 3) as well as live cell imaging of EB1-decorated microtubules in the same species (Strickland et al., 2005) support the notion that at the metaphase–anaphase transition, the asters have yet to contact the cortex. Once the asters contact the cell cortex, Ser19 phosphorylation becomes restricted to the cleavage plane (Figure 3), and staining of phospho-Ser19 myosin regulatory light chain (PMLC) and myosin II (Figure 4) suggests that this zone of Ser19-phosphorylated regulatory light chain reflects local regulation of myosin II activity, and not simple redistribution of the motor. Indeed, recent mapping studies of RhoGTPase activity in sea urchin eggs revealed a microtubule-dependent zone of RhoA activity that precedes cleavage furrow formation (Bement et al., 2005), and the equatorial band of Ser19-MLC may be a reflection of local activation of RhoA.
Our findings that myosin II activation was independent of astral microtubules were further explored by altering microtubule polymerization or organization (Figure 7). Although nocodazole did not significantly alter myosin II activation, the dynamics of the contractile response was altered (Figure 7B'). In control cells (Figure 7, A and A'), an increase in cortical contractility during this transition was followed by a brief relaxation before furrowing. In contrast, nocodazole-treated cells (Figure 7C') underwent a sharp increase in contractility that was sustained for up to 5 min without a subsequent relaxation phase. This raises the possibility that microtubules may partially suppress the global activation of contractility after anaphase onset. Similarly, whereas the presence of supernumery asters did not inhibit the global increase in contractility (Figure 7D'), the amplitude was dampened relative to controls. Together, these results suggest that although the initial myosin II activation is both independent of and occurs before cleavage plane determination, microtubules may play both positive and negative roles in confining contractility to the equatorial zone.
Signaling Pathways Regulating Myosin II Activation
Mapping of contractile activity by compression revealed that myosin II activation coincides with the metaphase–anaphase transition (Figure 2). This stands in contrast to earlier models for the timing of cytokinesis where myosin II activity was thought to be dependent on complete inactivation of cyclin-dependent kinase (Satterwhite et al., 1992; Satterwhite and Pollard, 1992). However, previous manipulation studies in sea urchin eggs (Shuster and Burgess, 1999) as well as in tissue culture cells (Rosenblatt et al., 2004) argue that myosin II activity may not be significantly suppressed during mitosis. Furthermore, mobilization of calcium early in mitosis resulted in a cortical contractile response (Figure 10), providing yet further evidence that myosin II is capable of responding to contractile signals in the presence of elevated cyclin-dependent kinase (CDK)1 activity. Studies of cylindrical sea urchin eggs suggested the presence of a molecular switch that signals the onset of cytokinesis and which is activated at the metaphase–anaphase transition (Shuster and Burgess, 2002b). It is possible that this switch identified in earlier experiments is related to the rise in cortical contractility observed in our compression assays.
There is strong evidence supporting the importance of ROCK as well as MLCK during cytokinesis (Poperechnaya et al., 2000; Matsumura et al., 2001; Chew et al., 2002; Matsumura, 2005). However, we find that although both signaling mechanisms are required for cytokinesis, they are required at different times. MLCK seems to be required for the global activation of myosin II at the metaphase–anaphase transition (Figure 8, C and D) as well as the ingression of the cleavage furrow (Figure 9), whereas inhibition of ROCK had no effect on the initial increase in myosin II activity, but it was required for furrow ingression (Figure 9). The notion that ROCK is required for myosin II regulation at later stages of cytokinesis is supported by time-lapse imaging of spherical ROCK-inhibited eggs, where furrows initiated but failed (our unpublished data). Together, these findings suggest that ROCK and MLCK are both required for cytokinesis but may serve different functional roles and may be activated at different times during mitotic progression. Indeed, studies in Drosophila indicate that although contractile rings can assemble and partially function in cells expressing nondegradable cyclin B3 (Parry and O’Farrell, 2001), Rho (and thus ROCK) function is not properly activated (Parry et al., 2003). Thus, Rho-dependent functions may lie downstream of the initial signal for cytokinesis, after microtubule contract with the cell cortex (Bement et al., 2005).
Global activation of contractility has been proposed in a number of models for cytokinesis (White and Borisy, 1983), but few mechanisms have been put forth to explain how a global activation of myosin II contributes to a process predicated on asymmetric distribution of force (cytokinesis). Schroeder proposed that a global activation of contractility primes the cortical cytoskeleton before cleavage plane determination and that astral microtubules disrupt this isometric contraction (Schroeder, 1981, 1990). Cytokinesis in Dictyostelium has been described as a two-phase process: an initial phase in which the cell transitions to a cylindrical shape and a second thinning phase during which the contractile ring constricts (Robinson et al., 2002; Reichl et al., 2005; Zhang and Robinson, 2005). It is during phase I of cytokinesis that the equatorial concentration of myosin II is at its greatest (Robinson et al., 2002), suggesting that the transition from a sphere to a cylinder requires the most force generation. It is possible that the principle role of MLCK is to provide contractile force during the initial steps in cell shape change (sphere to cylinder transition). This idea is supported by studies in isolated stress fibers, where MLCK is required for rapid contractile responses, and ROCK is required for slower, sustained contractions (Katoh et al., 2001). Last, myosin II regulation is spatially distinct in fibroblasts, with MLCK regulating peripheral contractility and ROCK regulating contractility in the cell center (Totsukawa et al., 2000; Totsukawa et al., 2004). Thus, it is not unreasonable to extrapolate such a model to sea urchin eggs, where MLCK regulates global (peripheral) contractile responses, and Rho-dependent kinases regulate contractility in response to spatial cues from the mitotic apparatus.
Reordering the Early Events of Cytokinesis
As discussed above, cytokinesis in animal cells is thought to include phases such as cleavage plane determination, contractile ring assembly, myosin II activation, ring constriction, disassembly of the contractile ring, and new membrane addition, followed by midbody formation. However, the finding that myosin II is activated before cleavage plane determination suggests that the above-mentioned events require some reorganization. Previous models for the timing of cytokinesis required that CDK1 activity drop before myosin II activation (Satterwhite et al., 1992). However, both our live cell analyses (Figures 1 and 2) as well as phospho-Ser19 staining (Figure 3) clearly demonstrate that myosin II activation occurs at the metaphase–anaphase transition, well before the other manifestations of mitotic exit. Myosin II contractility at the metaphase–anaphase transition both precedes and is independent of microtubules, thus placing activation of contractility before cleavage plane determination in the ordered phases of cytokinesis. In our revised model, the metaphase–anaphase transition triggers or is activated by a calcium transient (Groigno and Whitaker, 1998) that activates calmodulin and MLCK, thus resulting in myosin II activation and a global increase in cortical contractility. As CDK1 activity decreases and the astral microtubules elongate and contact the cortex, the global increase in contractility is transiently suppressed, resulting in a temporary decrease in contractility and relaxation of the cortex. Microtubule contacts with the cortical actin cytoskeleton directs the localized activation of Rho (Bement et al., 2005) and the recruitment of membrane microdomains (Ng et al., 2005), contributing not only to the organization of the ring but also to the maintenance of myosin II activation via ROCK and myosin phosphatase as the furrow ingresses. A detailed dissection of the relationship between mitotic exit and MLCK activation, similar to studies performed on cyclin destruction in Drosophila (Parry and O’Farrell, 2001; Echard and O’Farrell, 2003) as well as a functional evaluation of the apparent redundancy of myosin II-activating signals will provide better temporal resolution of this model.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-02-0119) on July 12, 2006.
Address correspondence to: Charles Shuster (cshuster{at}nmsu.edu)
Abbreviations used: CaFSW, calcium-free seawater; IP3, inositol-1,4,5-triphosphate; MLCK, myosin light chain kinase; MRLC, myosin regulatory light chain; NEB, nuclear envelope breakdown; ROCK, Rho-kinase
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0.007.
-tubulin (A, D, G, and J) and phospho-Ser19 MRLC (B, E, H, and K) localization and imaged by confocal microscopy. In the low magnification image shown in A–C (bar, 50 µm), approximately half of cells fixed late in metaphase had detectable phosphorylated regulatory light chain in the cortex. In contrast to metaphase cells (A–F), cells in anaphase with elongated astral microtubules had little light chain phosphorylation in the cortex except at the equator (G–I). By the onset of furrowing (J–L), phosphorylated light chain was concentrated in a broad band at the equator. Bar, 50 µm.
