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Vol. 19, Issue 1, 318-326, January 2008
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University of Massachusetts Medical School, Department of Physiology, Worcester, MA 01605
Submitted August 14, 2007;
Revised September 21, 2007;
Accepted October 17, 2007
Monitoring Editor: Fred Chang
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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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), it is generally agreed that the organization of actin and myosin filaments along the equator is related to equatorial contractility and cortical ingression. There are two prevailing hypotheses on how myosin and actin are recruited to the equatorial cortex. The cortical flow hypothesis proposes that myosin and actin from the polar cortex flow actively or passively into the equatorial cortex, as a consequence of either directed transport or differential cortical contractions (White and Borisy, 1983; Bray and White, 1988). The structural synthesis hypothesis argues that direct recruitment of molecules or small polymeric building blocks from the cytoplasm is responsible for the formation of the acto-myosin equatorial band (Pelham and Chang, 2001; Wu et al., 2006).
There is experimental evidence for the involvement of both cortical flow and direct structural synthesis in cytokinesis. The former was supported primarily by imaging membrane-bound beads (Wang et al., 1994), fluorescently labeled actin (Cao and Wang, 1990) or myosin (DeBiasio et al., 1996) in systems from live mammalian cells to Dictyostelium. In addition, several theoretical models predicted a concerted flow of cortical actin and myosin into the equator (White and Borisy, 1983; He and Dembo, 1997), as a consequence of global force balance over the cortex. Direct structural synthesis was supported by the appearance and/or growth of punctate acto-myosin structures in dividing frog embryos (Noguchi and Mabuchi, 2001), and yeast (Wu et al., 2006) and by the dependence of cytokinesis on genes that regulate de novo structural assembly (Gunsalus et al., 1995; Pelham and Chang, 2001; Severson et al., 2002). Although cortical flow and structural synthesis hypotheses are not mutually exclusive, their contributions to the recruitment of equatorial actin and myosin remain unclear. As several hypothetical mechanisms of cytokinesis make strong predictions about how equatorial structures are formed (Burgess and Chang, 2005), detailed analyses of cortical dynamics during early cytokinesis are likely to provide useful new insights.
The limitation to the current knowledge is in part due to the lack of high-resolution images that depict the assembly of equatorial cortex immediately before and during cytokinesis. Because of the strong signals from the cytoplasm, it has been very difficult to observe directly the assembly process in live cells using conventional epifluorescence optics. With its very short depth of field (<200 nm) and dramatically reduced background, the total internal reflection fluorescence microscopy (TIRF-M) is uniquely suitable for providing high-resolution images of structural dynamics on the cell cortex (Axelrod, 2001). In this study, we have combined TIRF-M with spatial temporal image correlation spectroscopy (STICS; Hebert et al., 2005), an approach similar to fluorescence speckle microscopy for precise tracking of the movement of structural inhomogeneities, and temporal differential microscopy (TDM), a novel image analysis method that measures the local kinetics of structural assembly or disassembly. Our observations lead us to the conclusion that actin and myosin follow distinct pathways to the equatorial cortex. Cortical flow is limited to actin and is not solely responsible for its equatorial concentration, whereas myosin is recruited directly from the cytoplasm. In addition, we show that the formation of equatorial myosin band involves the regulation of both assembly and disassembly activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drug Treatment and Microinjection
The drug concentrations of Y-27632 (Calbiochem, San Diego, CA), (–)-blebbistatin (Toronto Research, Toronto, ON, Canada), (+)-blebbistatin (Calbiochem, San Diego, CA), ML-7 (Calbiochem, San Diego, CA), latrunculin B (Calbiochem) were 40, 100, 100, 50, and 10–20 µM, respectively. For drug experiments, NRK cells at prometaphase were treated for at least 10 min before anaphase onset and the drug was maintained during the following period of imaging. C3 transferase (Calbiochem) was microinjected at a concentration of 0.5 mg/ml with fluorescein dextran as a marker (Molecular Probes, Eugene, OR). Injection of fluorescein dextran alone had no effect on mitosis or cytokinesis.
Immunofluorescence
For myosin II staining, cells were fixed in 1% formaldehyde, 0.1% glutaraldehyde, and 0.3% Triton X-100 for 1 min, followed by postfixation in 0.5% glutaraldehyde for 10 min. Myosin IIA was immunostained with anti-nonmuscle myosin IIA polyclonal antibodies (Covance Research, Princeton, NJ) at a dilution of 1:100. To stain for actin filaments, cells were fixed in 4% formaldehyde and 0.2% Triton X-100 for 10 min and incubated with Alexa-488-phalloidin or Alexa-546-phalloidin following the manufacturer's protocol (Molecular Probes).
Microscopy and Data Collection
Images were collected with either a Zeiss Axiovert-10 or Axiovert-200M inverted microscope (Thornwood, NY), equipped with a 100x, NA 1.30 Phase Plan-Neofluar objective lens for conventional epifluorescence optics, and a 100x, NA 1.45 
Plan-Fluar objective lens for TIRF-M. Light for TIRF-M was generated by a Lexel Model 94, 2 W argon ion laser operating at 10 mW, 488 nm for GFP proteins, or a 2.5 mW 543 nm HeNe laser for mCherry-actin (model LHGR-0200, Research Electro Optics, Boulder, CO). The laser beam was expanded with a beam expander and directed into the microscope using a focusing lens and several steering mirrors, adjusted such that the beam exits the objective lens as a parallel beam at an angle exceeding the critical angle for total internal reflection. Fluorescence images were collected with a cooled CCD camera (model NTE/CCD-512-EBFT, Princeton Instrument, Trenton, NJ, or Model DV-887-DCS-BV, Andor Technology, Belfast, United Kingdom), with 0.5- or 1-s exposure and subtracted with a dark-count image. Except for linear adjustments across the entire image to optimize the display, the images were unaltered. Data acquisition and analyses were performed with a combination of custom software, ImageJ (NIH; http://rsb.info.nih.gov/ij/) and Excel (Microsoft, Redmond, WA).
Image Analysis
Standard image analysis procedures, including kymograph analysis and intensity integration, were performed with a combination of custom software, ImageJ (NIH) and Excel (Microsoft). STICS was implemented as custom software, using a similar cross-correlation approach as described previously (Hebert et al., 2005; Ji and Danuser, 2005). Each set of displacement vectors was generated by averaging displacements detected in a set of five consecutive frames taken over 
12 s. The detection window for movements was set at 1.3 x 1.3 µm2 as the default and adjusted adaptively as described by Ji and Danuser (2005). TDM was also implemented as custom software. It involved pixelwise computation of the intensity differential, (It+
t – It)/(It+t + It)/t/2, where It is the average intensity around the pixel at time t and t = 4–7 s. This quantity equals approximately the local rate of percentage change in intensity. To control the noise some images were filtered with a median filter before the computation. For visualization, the resulting positive or negative values were scaled linearly and rendered in pseudocolor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Maupin et al., 1994), rather than single molecules. Cortical actin on dividing cells consisted of dots, patches, short fibers, and diffuse structures (Figure 1A).
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60 and 100 s after chromosome segregation, respectively (Figure 1B), suggesting that they may follow different pathways.
Myosin Shows No Detectable Cortical Flow in Early Cytokinesis
To determine if the assembly process of equatorial myosin involves cortical flow, as has been suggested previously (White and Borisy, 1983), we collected time-lapse TIRF-M movies of dividing cells expressing GFP-myosin. Direct observations of cortical myosin in more than 20 cells provided no indication of directional movement toward the equator during early equatorial assembly (Figure 2A, Supplementary Video 1). Moreover, kymograph analysis showed only horizontal tracks indicating that there was no long-range movement of myosin throughout the cortex along any directions (Figure 2B). Consistent results were obtained with STICS, which tracked the movement of features within 1.3 x 1.3 µm2 regions (Figure 2C). The increase in equatorial myosin concentration appeared to involve a combination of an increase in the intensity of pre-existing myosin structures and de novo appearance of new myosin dots along the equator (Figure 2A and 2B d, e, boxed areas), which together lead to a 45 ± 12% increase in average intensity along the equator during a 30-s interval in early cytokinesis. The flanking regions showed a concomitant intensity decrease of 14 ± 6% (Figure 2D), suggesting changes in the relative association and dissociation kinetics between the equatorial region and the rest of the cortex.
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t – It)/(It+t + It)/t/2, with pairs of images It, It+t separated temporally by t. This quantity approximates the rate of percentage change in intensity. The resulting images were rendered in color, with bright orange marking regions of strong proportional increases in intensity and bright blue marking regions of strong decrease (n = 31; Figure 3A, and Supplementary Video 2). TDM revealed strong assembly domains of myosin up to 10 µm in diameter throughout the cortex. These domains lasted typically 15–20 s, during which some of them traveled across the cortex (Figure 3A). These observations indicate that myosin assembly was not confined to the equatorial region (Lucero et al., 2006), although the equatorial region may have a higher net assembly activity than elsewhere.
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Cortical Myosin Is Affected in Different Ways by Small GTPase Rho, Rho Kinase, Myosin Light-Chain Kinase, and Its Own ATPase Activities during Cytokinesis
Cortical dynamics of myosin is likely affected by its ATPase and self-assembly activities, which are in turn regulated by the phosphorylation of myosin regulatory light chain (MRLC) by a number of kinases including the myosin light-chain kinase (MLCK) and Rho-dependent kinase (ROK; Bresnick, 1999; Poperechnaya et al., 2000; Fukata et al., 2001; Chew et al., 2002; Matsumura, 2005). Y-27632, an inhibitor of ROK, delayed cytokinesis as reported previously (Kosako et al., 2000). The formation of equatorial myosin band appeared uninhibited (Figure 4A). However, time-lapse movies and kymographs indicated that equatorial myosin dots were surprisingly stable in Y-27532–treated cells (Figure 4A). Myosin dots outside the equator became sparse and highly transient compare with those in control cells shown in Figure 2, suggesting that they failed to associate stably with the cortex (Figure 4A; Supplementary Video 3). In addition, TDM analysis showed a strong inhibition of dynamic myosin domains throughout the cortex (Figure 4D). Injection of C3 transferase (a Rho inhibitor), unlike Y-27632 treatment, caused complete inhibition of the recruitment of myosin dots along the equator (Figure 4C, Supplementary Figure 2; (O'Connell et al., 1999; Kamijo et al., 2006), suggesting that ROK is not the only effector of Rho that regulates equatorial myosin recruitment. ML-7, an inhibitor of MLCK, caused a delay in cytokinesis without an apparent effect on cortical myosin dots along or outside the equator (Supplementary Video 4).
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; Dean et al., 2005; Kamijo et al., 2006), which induces depolymerization of actin filaments and thereby inhibits the actomyosin ATPase. This was also the case for NRK cells (Supplementary Figure 3, and Supplementary Video 6). However, the equatorial myosin band gradually widened over 20 min after anaphase onset, suggesting that acto-myosin interactions play a role in restricting the equatorial myosin localization (Supplementary Figure 3).
Recruitment of Equatorial Actin Involves a Combination of Myosin Motor-dependent Fluxes and De Novo Assembly
The delay of equatorial actin recruitment relative to that of myosin suggested that actin and myosin may follow different pathways (Figure 1B). To observe directly the equatorial recruitment of actin, we collected time-lapse movies of dividing cells expressing GFP-actin or mCherry-actin. In contrast to myosin, actin filaments showed a striking flux toward the equator during cytokinesis (observed in 20 cells; Figure 5, A and B, Supplementary Videos 7 and 8), at an estimated average speed of 2.1 ± 0.6 µm/min based on the slopes of kymographs (Figure 6B). The flux was unlikely to reflect the movement of the entire cortical actin structure, because some actin structures moved to the equator, whereas others remained stationary (Figure 5B). In addition, small actin structures were observed to dissociate from large patches and move into the furrow, suggesting that actin disassembly or severing, rather than a global collapse of the cortical network, may be involved in the generation of the flux (Supplementary Video 7). Moreover, STICS analysis indicated heterogeneous speeds and movement patterns, and juxtaposition of directional and random movements (Figure 5C), further arguing against global movement of a cross-linked cortex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), it is generally believed that the recruitment process holds an important key to the understanding of the mechanism of cytokinesis. For example, the polar relaxation hypothesis predicts that the entire cortical structures should move toward the equator, due to the weak forces in the polar region relative to the strong contractions along the equator (White and Borisy, 1983). In contrast, some versions of the equatorial contraction mechanism emphasize a de novo mechanism for equatorial structural assembly (Wu et al., 2006), whereas other versions incorporate cortical flow as part of an assembly process (He and Dembo, 1997). However, because of the lack of high-resolution images depicting the recruitment process in mammalian cells, there has been no definitive evidence for these proposed mechanisms. The clarity of images provided by TIRF-M has allowed us to determine unambiguously the behavior of cortical actin and myosin during early cytokinesis.
Equatorial Recruitment of Myosin in Early Cytokinesis Does Not Involve Cortical Flow
Although dot-like structures of cortical myosin have been observed during cytokinesis in fixed samples by confocal microscope (Maupin et al., 1994), little is known about how they become concentrated along the equator. Our results, showing no detectable, long-range flow of myosin dots toward the equator, argued against the cortical flow hypothesis for myosin recruitment and supported either the assembly of myosin minifilaments from cytoplasmic subunits or recruitment of preassembled minifilaments. Although the polar cortex showed a net loss of myosin dots, myosin appeared to be released into cytoplasm first and recruited to the equator. This shift in the balance of myosin cortical association may involve differential interactions of polar and equatorial cortices with astral microtubules (Werner et al., 2007).
Our results appear to contradict previously reported fluxes of myosin in mammalian cells (DeBiasio et al., 1996), and in Dictyostelium (Yumura, 2001). However, the present observations were focused on early assembly events of equatorial cortex. During the subsequent phase of active ingression, we did observe some limited movements of myosin dots on the equatorial cortex and in the immediately adjacent region, which likely reflect contractile activities and may explain previous observations of myosin movements (DeBiasio et al., 1996). In addition, different organisms may adopt different mechanisms of myosin recruitment, as suggested by the multiple mechanisms of cytokinesis in motile Dictyostelium cells (Uyeda et al., 2004).
Equatorial Recruitment of Actin Involves a Combination of Cortical Flow and De Novo Assembly
In contrast to myosin, cortical actin showed a striking flux toward the equator, consistent with previous observations at a limited resolution using conventional optics and microinjected fluorescent phalloidin (Cao and Wang, 1990). In addition, the flux took place in discrete domains flanking the equator, and at least part of the moving structures appeared to be generated by severing and/or disassembling existing actin structures, suggesting that the flux was an active, selective process rather than global cortex movement as a result of force balance.
Treatment of blebbistatin abolished the actin flux and inhibited actin turnover, but did not inhibit actin concentration in the equator (Figure 7; Guha et al., 2005; Murthy and Wadsworth, 2005), suggesting that the flux was driven by myosin motor activities and that there were additional, flux-independent mechanisms, likely de novo assembly as suggested in other systems (Noguchi and Mabuchi, 2001; Pelham and Chang, 2002; Severson et al., 2002; Wu et al., 2006). Myosin may be involved in both processes, by providing forces for the flux and by facilitating the turnover of actin cortex to generate the sources of flux. Because blebbistatin also completely inhibited furrow ingression, it is possible that cytokinesis requires not only an equatorial acto-myosin band but also actin flux and/or turnover. In addition, although the major events of cytokinesis take place along the equator, the complex picture presented by TIRF-M suggested that the process may involve the entire cortex.
Equatorial Myosin Recruitment Involves the Regulation of Both Assembly and Disassembly Processes
Dynamic cytoskeletal structures are maintained by a balance between assembly and disassembly activities. Previous immunostaining studies have shown a concentration of mono- and di-phosphorylated MRLC along the equator, which is known to activate the myosin ATPase and promote the assembly of filamentous myosin structures (Matsumura et al., 1998, 2001;). These observations were often interpreted as suggesting an increase in the rate of myosin assembly or cortical association along the equator. However, equatorial MRLC phosphorylation and myosin structures, may be stimulated equally effectively by decreasing the rate of disassembly or dissociation from the equatorial cortex, as was suggested by the requirement of regulated myosin phosphatase activities in cytokinesis (Matsumura, 2005).
Time-lapse TIRF-M and TDM revealed surprisingly active domains of cortical myosin assembly/association throughout the cell cortex immediately after anaphase onset. These domains were not detected in previous studies, because without TIRF-M, their contrast against the strong cytoplasmic signals was likely to be very low. In addition, the transient, random nature of these domains likely made them very difficult to notice in fixed cells. The global presence of these assembly domains suggested that assembly signals for myosin were not confined to the equatorial region (Lucero et al., 2006).
Previous studies showed that the primary function of ROK during cytokines is to elevate the phosphorylation of MRLC (Dean and Spudich, 2006). The strong inhibition of dynamic domains of cortical myosin assembly by Y-27632, as shown in the present study, suggested that ROK and ROK-mediated phosphorylation of MRLC were involved in the recruitment of myosin throughout the cortex. However, the presence of equatorial myosin band despite the inhibition of ROK suggested a second, ROK-independent pathway that directly recruited myosin to the equatorial cortex.
TDM analysis further revealed local inhibition of myosin disassembly/dissociation along the equator, which likely contributed to the build up of myosin concentration. Unexpectedly, the stability of equatorial myosin in the presence of Y-27632 suggested that the stabilization involved a mechanism independent of ROK-induced direct MRLC phosphorylation or inhibition of myosin phosphatase (Fukata et al., 2001), whereas the turnover was dependent on ROK. Furthermore, actin filaments may play a role in the stability of myosin, as suggested by the gradual expansion of the equatorial myosin band in latrunculin-treated cells. Other molecules such as anillin have also been implicated in the recruitment of myosin, possibly by stabilizing myosin association along the equator (Straight et al., 2005).
The effects of blebbistatin on cortical myosin distribution and dynamics were strikingly different from those of Y-27632 or C3. Inhibition of myosin ATPase activity by blebbistatin appeared to inhibit cortical myosin disassembly/dissociation without affecting cortical myosin assembly/association, indicating that the ATPase activity is not required for its cortical association but may be required for its turnover. In contrast, C3caused strong inhibition of equatorial myosin recruitment, suggesting that the primary effect of Rho on myosin was not the regulation of ATPase activities, but possibly its self assembly, disassembly, and/or cortical association.
In conclusion, many previous models of cortical ingression involved global, coupled movements of actin and myosin filaments into the equatorial region. The present study showed that, while a prominent myosin-dependent actin flux did occur during the cytokinesis of NRK cells, the movement was by no means global and no such movement was detectable for myosin. In addition, the formation of the equatorial actin band involved a second, myosin-independent, de novo assembly process. These results support an active mechanism that promotes structural organization at the equator, rather than a passive mechanism as a consequence of differential cortical contractile activities. Moreover, the discovery of dynamic domains of myosin assembly throughout the cortex, combined with a localized suppression of myosin disassembly/dissociation along the equator, suggests that the spatial and temporal control of cytokinesis may involve the regulation of both assembly and disassembly activities.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Yu-Li Wang (yuli.wang{at}umassmed.edu)
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