Article |
Address correspondence to Craig A. Mandato, Department of Zoology, University of Wisconsin, 1117 West Johnson St., Madison, WI 53706. Tel.: (608) 262-5683. Fax: (608) 262-9083. E-mail: camandato{at}facstaff.wisc.edu
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Abstract |
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Key Words: wound healing; cytokinesis; actin polymerization; dorsal closure; myosin polymerization
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Introduction |
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While each of these structures plays a different role and forms in response to different stimuli, they share in common the properties of flexibility and transience. That is, actomyosin rapidly accumulates at the rear of cells in response to polarizing stimuli and reorganizes when the cell stops moving (e.g., Moores et al., 1996; Verkhovsky et al., 1999). In addition, the cytokinetic apparatus can be repositioned by experimental displacement of the spindle (Rappaport, 1996), whereas rings of F-actin and myosin 2 assemble in amphibian oocytes wherever the cell is wounded and disassemble after the completion of healing (Bement et al., 1999).
How does the cell assemble structures that are both sufficiently robust to drive contraction and sufficiently dynamic to respond to changing stimuli? Because the cortex of most cells is comprised of an interconnected network of F-actin and myosin 2, localized contractile structures can in principle be generated as the result of any signal that generates local contraction. Specifically, an initial, localized increase in actomyosin-based contraction is expected to be amplified by the recruitment of actomyosin from adjacent regions due to cortical flow. Cortical flow is the translocation of cortical F-actin, myosin 2, and cell surface proteins parallel to the plane of the plasma membrane (Bray and White, 1988). Such flow is driven by contraction itself, with the integrated cortical cytoskeleton pulling more actomyosin to the site of initial contraction which, in turn, results in greater contractility in that region. Consistent with this notion, cortical flow has been observed during cytokinesis (e.g., Cao and Wang, 1990), pseudocleavage in Caenorhabditis elegans (Hird and White, 1993), cell locomotion (e.g., DeBiasio et al., 1996), and experimentally induced contraction in Xenopus oocytes (e.g., Benink et al., 2000).
However, several observations are inconsistent with contraction-driven cortical flow being the only mechanism for recruitment of F-actin or myosin 2 to nascent contractile structures. For example, myosin 2 lacking motor activity localizes in the prospective cleavage furrow in Dictyostelium (Yumura and Uyeda, 1997; Zang and Spudich, 1998). In addition, in both budding yeast and fission yeast, the initial localization of myosin 2 to the incipient contractile ring is not strictly dependent on F-actin and/or interaction of the myosin 2 with F-actin (e.g., Bi et al., 1998; Lippincott and Li, 1998; Naqvi et al., 1999; Motegi et al., 2000). Further, myosin 2 is found at oocyte wound borders even after disruption of F-actin (Bement et al., 1999).
The Xenopus oocyte system is particularly useful for experimental analyses of localized actomyosin recruitment, in that wounds can be produced on demand and in an orientation which facilitates imaging. Here we have analyzed assembly of actomyosin rings in living Xenopus oocytes using a combination of laser wounding, time-lapse confocal (four-dimensional [4D]*) microscopy, and experimental manipulation of the actomyosin cytoskeleton. The results reveal that cortical flow and contraction work in concert with de novo assembly of F-actin and myosin 2 to establish and close woundinduced actomyosin arrays.
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Results |
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Wound-induced actomyosin rings are contractile
If wound closure is based on contraction, two simple predictions follow. First, square wounds should round up as tension is exerted around the wound. Second, elongate wounds should shorten fastest along their long axes. 4D analysis of wound closure confirmed both of these predictions: square wounds produced by the imaging laser rounded up before closure (Fig. 1
A, and Video 1), and oval wounds produced by two rapid, adjacent pulses from the nitrogen dye pump laser closed most rapidly along their long axes until the wound became circular (Fig. 1 B), regardless of the probe used for imaging (Fig. 1 C).
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The above experiments were limited by the fact that after rewounding, 1 min elapsed before imaging resumed (the time required to reconfigure the microscope from a Z-scan to en face imaging). Thus, we were unable to follow the opening of the ring immediately after damage. Further, because wounding itself generates a response, it is impossible to determine to what extent the observed effects of the rewound resulted from previously existing tension versus a response to the rewound. Therefore, we developed a complementary strategy in which wounds were "cauterized" by extending the duration of the laser scan used to create wounds. This produced roughly square regions of burnt cytoplasm within the wound that acted as barriers (Fig. 1 E). Upon contact with the cauterized squares, the ring edges usually either ceased advancement, moved up and over the square, or dove below the square. However, the rings occasionally stretched and broke after contacting the burnt cytoplasm (Fig. 1 E, and Video 2). In such cases, the broken edges of the rings recoiled laterally around the wound toward the unbroken portion, exactly as expected if the ring itself were under circumferential tension. Again, the recoil of the broken edges was limited to 1020 µm, and the rest of the ring, after a brief (3060 s) pause, continued to close, implying that the ring is tightly anchored at many points along its circumference.
Cortical flow creates a vortex of recruitment of stable F-actin to wounds
We next sought to characterize patterns of movement of stable (i.e., previously assembled) F-actin during the wound healing process, since cortical flow is expected to act on stable F-actin. To this end, oocytes were injected with AX-phalloidin, wounded, and imaged. Particle tracking and kymograph analysis of 4D videos showed that stable F-actin flowed toward wounds from cortical regions around the wound edge at 3 µm/min (Table I), and accumulated in a region of high F-actin density bordering the wound (Fig. 2
A, and Video 3). The flow resulted in the formation of a characteristic dark halo of F-actin depletion around the wound that moved away from the wound over time at a mean rate of 1.5 µm/min (Table I). Flow and halo spreading were independent of the forward movement of the wound edge (data not shown).
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De novo polymerization of actin around wound borders
To characterize contributions of de novo polymerization to contractile ring assembly, oocytes were injected with OG-actin, wounded, and analyzed by 4D microscopy. After assembling a ring of actin around wounds, OG-injected, wounded oocytes displayed perpendicular F-actin cables which flowed toward wound borders, as well as a dark halo of actin depletion around the wound, consistent with results obtained from AX-phalloidininjected oocytes (Fig. 3
A, and Video 5). Fluorescence intensity measurements revealed the dark halo of the F-actin signal around wounds as a trough between the "background" signal at regions distal to the wounds and the peak signal bordering the wound (Fig. 3 B). Comparison of intensity scans taken at increasing times after wounding demonstrated that at early time points (before those shown in Fig. 3 A), the trough was minimal or absent, but it became quite pronounced at later points, showing that accumulation of F-actin at wound borders precedes the onset of cortical flow.
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To characterize the spatial relationship between stable and dynamic F-actin, two additional experiments were conducted. First, oocytes were injected with TR-phalloidin as a marker for stable F-actin and then OG-actin as a probe for dynamic F-actin. After wounding, the stable F-actin was localized as a narrow ring around wounds within a broader ring composed of dynamic F-actin (Fig. 4 A). Likewise, double labeling with OG-actin and TMRmyosin 2 revealed that the myosin 2 was focused as a narrow ring within a broader ring of dynamic F-actin (Fig. 4 A). While the width of the zone of dynamic F-actin increased over time, the width of the contractile actomyosin ring remained constant (Fig. 4 B).
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Myosin 2 accumulates around wounds with only limited recruitment from the cortex
To monitor recruitment of myosin 2 to wound borders in vivo, oocytes were injected with TMRmyosin 2, and then wounded and analyzed as above. After wounding, TMRmyosin 2 rapidly accumulates around wound borders in bright foci that fuse to form a continuous, circumferential ring that closes with time (Fig. 5
A, and Video 9). Analysis of myosin 2 dynamics revealed two principle differences relative to actin: myosin 2 flow was much less obvious than F-actin flow, and myosin 2 was present in regions bordering the wound as punctae rather than continuous cables (Fig. 5 A, and Video 9). The lesser degree of flow was also apparent as reduced myosin 2 depletion in wound-flanking regions detected by fluorescence intensity measurements of both living (Fig. 5 B) and fixed samples (Fig. 5 C).
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A zone of actin polymerization around wound borders
The foregoing results suggested that both F-actin and myosin 2 can accumulate around wounds by mechanisms independent of contraction-dependent cortical flow. To test this point directly for F-actin, oocytes were subjected to treatments designed to prevent cortical flow and/or contraction. Cortical flow can be prevented by incubation of oocytes in high concentrations of lectins, such as wheat germ agglutinin (WGA), which crosslinks cell surface proteins, thereby immobilizing the cortex (Canman and Bement, 1997). In WGA-treated oocytes, wound-induced cortical flow was inhibited, no dark halo formed, and wounds failed to round up and close (Fig. 6, A and B)
. Nevertheless, F-actin still accumulated in a zone around wounds in WGA-treated oocytes, although the zone was unstable (Fig. 6 A). As an alternative approach, oocytes were injected with N-ethylmaleimide (NEM)-treated S1, which binds specifically and irreversibly to the myosin binding site of F-actin, and thereby inhibits contractility (Meeusen and Cande, 1979). NEM-S1 inhibited cortical flow, wound rounding up, and wound closure; however, as with WGA, actin accumulated around wound borders (Fig. 6, A and B). The actin accumulation was unstable and eventually disappeared from the wound border (Fig. 6 A).
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Uncoupling of myosin 2 recruitment from cortical flow and contraction
To test whether myosin 2 recruitment to wound borders requires cortical flow or contractility, oocytes were injected with TMRmyosin 2, treated with WGA to block cortical flow, wounded, and then analyzed by 4D microscopy. As with actin (see above), WGA treatment failed to prevent the accumulation of TMRmyosin 2 to wound borders, although it failed to coalesce into a tight circumferential array (Fig. 7
A). As an alternative means of preventing contraction and cortical flow, TMR-myosin 2injected oocytes were treated with cytochalasin and then wounded. After wounding, myosin 2 accumulated around the wound border in bright foci which failed to coalesce in a continuous ring (Fig. 7 B). Higher magnification videos revealed that such foci formed and grew progressively larger in the absence of any apparent recruitment from cortical regions (Fig. 7 C, and Video 11).
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Breakage of the contractile ring causes localized destabilization of the polymerization zone
The fact that actin and myosin 2 accumulated around wound borders when contraction and cortical flow were prevented demonstrated that neither contractility nor the contractile ring are required for formation of the polymerization zone. However, the fact that the zone eventually disappeared in the presence of NEM-S1 and WGA suggested that contractility, the contractile ring, or both help maintain the zone. To test this notion, oocytes were injected with both OG-actin and TMRmyosin 2, and then subjected to wound cauterization. The TMRmyosin 2 allowed identification of the contractile ring within the broader zone of polymerization revealed by OG-actin (Fig. 4 A). Upon contact with the cauterized region, one portion of the contractile ring ceases movement, becomes stretched, and then snaps (Fig. 8
and Video 12). The local stopping and stretching of the contractile ring was accompanied by local stopping and broadening of the polymerization zone (Fig. 8 and Video 12). Upon breakage of the contractile ring, the zone transiently disappeared and then reappeared as an arc extending off of the rest of the actomyosin array around the wound, but distal from the wound edge. Thus, the contractile ring is required for forward movement of the zone and to keep it focussed around the edge of the wound. The supplemental videos are available at http://www.jcb.org/content/vol154/issue4.
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Discussion |
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The second finding is that F-actin cables flowing toward wounds accelerate sharply as they leave regions of high cortical actin density. This implies the existence of a positive feedback loop in which flow-dependent depletion of F-actin promotes further, rapid recruitment of F-actin, which in turn results in additional depletion. This observation provides direct, empirical support for cortical flowdependent positive feedback in contractile processes, a longstanding, but previously untested notion (e.g., White and Borisy, 1983; Mandato et al., 2000). It also confirms the idea that spatially restricted destabilization of cortical actomyosin may be as important for localized contractile phenomena as contraction itself, an idea recurrent in both the cytokinesis (White and Borisy, 1983; Mandato et al., 2000) and cell locomotion (Taylor and Fechheimer, 1982; DeBiasio et al., 1996) literature.
The third finding, and the most surprising, is the formation of a restricted zone of de novo actin and myosin 2 assembly that works in concert with the classic contractile ring of continuous cables that run parallel to the wound border. Fluorescence intensity measurements indicate that de novo polymerization provides the initial signal for actomyosin accumulation around wound borders which, in turn, drives recruitment of F-actin via contraction and flow. Whether myosin 2 is also recruited via flow is unclear, since repeated attempts to document continuous flow failed. Instead, myosin 2 foci near the wound moved at the same rate as the wound edge, or in an intermittent manner. Even more surprisingly, myosin 2 foci often moved either perpendicular to or against the direction of flow. Thus, in addition to de novo polymerization, myosin 2 recruitment is mediated by a motility mechanism distinct from that used for F-actin. This result is not predicted by standard models of cortical flow, although we cannot rule out the possibility that myosin 2 that we cannot detect with our methods is also moving by a standard flow-based mechanism.
The results with the flow and contraction inhibitors indicate that, once formed, the polymerization zone is relatively unstable and requires contraction and flow for its maintenance as well as its forward movement. These results were confirmed by cauterization-induced ring breakage: stretching of the ring is accompanied by broadening of the zone, whereas its breakage results in transient disappearance of the zone. While it is easy to understand how contraction of the ring moves the wound edge forward, it is more difficult to understand why the ring is necessary for maintenance of the zone. One possibility is that the ring acts as a moveable scaffold for signaling molecules responsible for de novo assembly of actin and myosin 2.
An additional role for the zone of actomyosin assembly is demonstrated by the finding that late in the process of healing, F-actin fingers on opposing sides of the wound contact each other and pull the wound edges toward each other. The existence and dynamics of the F-actin fingers is remarkable for two reasons. First, in a detailed study of F-actin organization in fixed, cultured cells undergoing cytokinesis, Fishkind and Wang (1993) deduced the existence of ordered arrays of F-actin extending ahead of the cytoplasmic apparatus (see Fishkind and Wang, 1993, Fig. 9). Second, the behavior of the fingers is eerily reminiscent of ventral enclosure in C. elegans (Raich et al., 1999) and dorsal closure in Drosophila (Jacinto et al., 2000), wherein filapodia extend from and link opposing epithelial cells at the midline of the embryo. Consistent with our findings, Jacinto et al. (2000) found that in Drosophila this mechanism is operative primarily in the late stages of dorsal closure, and that the contacting filapodia exert force on the epithelial margin, as judged by its inward bending at points of contact (Jacinto et al., 2000). Obviously, the fingers differ from filapodia found in moving epithelia in that they are contained within a single cell. Nevertheless, their association with a contractile ring further supports the assertion that diverse, contractile ringdependent events may have conserved evolutionary roots (e.g., Bement et al., 1999; Woolley and Martin, 2000).
Whether a localized zone of actomyosin polymerization is also a component of other contractile structures remains to be seen, but this possibility could account for recent findings from Dictyostelium and yeast (see Introduction). The observations in fission yeast are especially striking in that they show discrete myosin 2 foci at the fission site in the absence of F-actin (Motegi et al., 2000). In addition, it was recently found that myosin 2 accumulates ahead of actin in cleavage furrows in Xenopus embryos (Noguchi and Mabuchi, 2001), suggesting that its recruitment may be F-actinindependent. Local regulation of myosin 2 and actin assembly during cytokinesis is also consistent with studies suggesting local control of myosin 2 regulatory light chain phosphorylation (e.g., DeBiasio et al., 1996; Matsumura et al., 1998; Poperechnaya et al., 2000) and the dependence of cytokinesis on proteins that regulate actin assembly and disassembly (e.g., Balasubramanian et al., 1994; Gunsalus et al., 1995), respectively.
What cellular and molecular mechanisms underlie the zone of actin and myosin 2 assembly? Three nonexclusive mechanisms are suggested by the literature: (a) Cdc42 is a likely candidate since the zone is the site of comet formation and comet formation is Cdc42-dependent in Xenopus egg extracts (e.g., Ma et al., 1998). (b) Likewise, PIP2 regulates F-actin comet formation in cultured cells (Rozelle et al., 2000), suggesting that this lipid might also be involved in actin dynamics in the polymerization zone. (c) Similarly, PKCs may act upstream of one or more of the rho class GTPases, since PKC agonists trigger formation of comets in Xenopus eggs (Taunton et al., 2000). Regardless of the upstream players, the polymerization zone is subject to very tight spatial control: neither F-actin nor myosin 2 spread far into the wound or far away from it. At the same time, the zone is resilient, disappearing and reappearing after stretching and breakage of the contractile ring, or rapidly assembling as an arc off of a severed ring.
In summary, the combined input of a contractile ring and a zone of actomyosin polymerization endow wound-induced actomyosin arrays with the strength, speed, and flexibility necessary for efficient assembly and closure. Therefore, it will be of interest to see if polymerization zones are associated with the contractile arrays that drive cytokinesis, morphogenesis, and multicellular wound healing. It will also be important to determine how the cell generates and controls such zones in response to wounding.
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Materials and methods |
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Laser wounding
Laser wounds were made through a 10 or 25x objective with either a Micropoint pulse nitrogenpumped dye laser (Laser Science, Inc.) or with a Krypton/Argon Bio-Rad Laboratories imaging laser (American Laser Corporation). Oocytes were placed between a glass slide and a coverslip separated by grease and positioned in the laser path. Wounding with the nitrogen laser, which emitted 120 uJ of 337 nm energy in 2-ns pulses (15.9 mW/cm2 at the cell surface) resulted in small, round wounds. Wounding with the imaging laser using both 488- and 586-nm laser lines at 100% power (3.7 mW/cm2 at the cell surface) produced square wounds.
4D . live Imaging
All 4D imaging was performed using a 100 M microscope (Axiovert; Zeiss) with the Bio-Rad 1024 Lasersharp Confocal software package. For each time interval, 912 1,024 x 1,024 images were collected with a Kalman averaging of 23. Step sizes were 0.361 µm. 4D videos were constructed from the confocal stacks with the Lasersharp software and imported into NIH Object Image v2.06 for analysis and kymograph construction. Further image analysis and processing were performed with Adobe Photoshop® 5.0, Metamorph 4.5 (Universal Imaging Corp.), Quicktime 4.1.2 (Apple Computer, Inc.) or Adobe Premiere® 5.1. Statistical analysis were performed using Microsoft Excel.
Perturbation of healing
To disrupt cortical flow and actomyosin array formation, oocytes injected with fluorescent probes were preincubated for 1 h in 40 µM cytochalasin B or 100 µg/ml WGA (Sigma-Aldrich) or injected with NEM-S1 at a needle concentration of 4 mg/ml immediately before wounding.
Confocal microscopy of fixed oocytes
After wounding, oocytes were fixed overnight in 40 mM Hepes, pH 7.6, 100 mM KCl, 3 mM MgCl2, 150 mM sucrose, 10 mM EGTA, 0.1% Triton X-100 containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 1 µm taxol. Oocytes were washed for 24 h in TBS containing 0.1% NP-40 (TBSN) and stained in TBSN plus 1 U/ml TR-X-phalloidin (Molecular Probes). After a 24 h wash in TBSN, samples were mounted and viewed. Serial optical sections were collected on a Bio Rad Laboratories 1024 laser-scanning confocal microscope. For double or triple labeling of F-actin, injected myosin 2, and endogenous myosin 2, TMR-myosininjected oocytes were wounded, fixed, and washed as above, and then stained for F-actin and for endogenous myosin 2 using an affinity-purified Xenopus myosin 2A antibody (provided by Dr. Bob Adelstein, National Institutes of Health, Bethesda, MD) as described previously (Bement et al., 1999). Profilin and arp3 antibodies (Welch et al., 1997) were a gift from Dr. Matt Welch (University of California, Berkley, CA) and were used for both immunoblotting and immunofluorescence. Immunofluorescence was as above; for immunoblotting, Xenopus egg extracts were separated on 4 20% SDS-acrylamide gels, transferred to nitrocellulose, immunoblotted with the -profilin and
-arp3 antibodies followed by HRP secondary antibodies, and developed using the ECL Western blotting system (Amersham Pharmacia Biotech).
Online supplemental material
All 4D confocal time-lapse videos were created using Bio Rad Laboratories LaserSharp software and produced in Quicktime 4.1.2 format. Videos are available at http://www.jcb.org/content/vol154/issue4.
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Footnotes |
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* Abbreviations used in this paper: AX, Alexa 488; 4D, four-dimensional; NEM, N-ethylmaleimide; OG, Oregon green; TMR, tetramethylrhodamine; TR, Texas red; WGA, wheat germ agglutinin.
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Acknowledgments |
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This work was supported by the National Institutes of Health (GM52932- 04A1); C.A. Mandato is supported by a Guyer Post-Doctoral Fellowship. The National Science Foundation supported acquisition of the confocal microscope used in this study (NSF9724515 to J. Pawley) and early phases of this project (MCB 9630860).
Submitted: 22 March 2001
Accepted: 10 July 2001
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