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R602 Dispatch

Wound healing: The power of the purse string Daniel P. Kiehart

Recently, Xenopus oocytes have been shown to repair process that mimics locomotion or invasion. These cells wounds using a contractile system composed of actin can also assemble a supracellular contractile ring or purse and myosin-II. The work underscores the importance of string composed of actin and myosin-II that contracts to actin-based myosin-II contractility in cellular and draw the wound margins inward. Together, these supracellular ‘purse strings’ that function in diverse processes contribute to most or all of the cell movements biological processes. that characterize epithelial wound healing.

Address: Department of Cell Biology, University Program in Genetics and University Program in Cell and Molecular Biology, Duke University In a recent paper, Bement et al. [9] focused their attention Medical Center, Durham, North Carolina 27710, USA. on the healing of wounds caused to the single-celled E-mail: [email protected] Xenopus oocyte. They showed that, in the oocyte, wound healing is Ca2+ dependent and involves an actomyosin-II Current Biology 1999, 9:R602–R605 http://biomednet.com/elecref/09609822009R0602 purse string that appears to drive ingression of the wound margins (Figure 1a). Within seconds of the injury, actin fil- © Elsevier Science Ltd ISSN 0960-9822 aments and myosin-II were recruited to a ring-like struc- ture at the margin of the wound. Interestingly, the rings of Throughout phylogeny, individual cells, complex tissues actin and myosin were not precisely coincident: myosin and the arrangements of tissues that comprise local was recruited to the innermost part of the ring, actin to the regions of whole organisms have the ability to ‘heal’ them- outermost part, with overlapping localization in the selves in response to physical insult. For single cells, the middle. Microtubules were also rapidly recruited to the main problem in the healing process is the rapid repair of site of the wound. Accumulation of actin, myosin-II and the plasma membrane [1]. Such remarkable healing microtubules began within seconds or tens of seconds and events ensure plasma membrane integrity and can even continued for minutes. Over the course of 10–15 minutes, occur in response to normal physiological activity, in the healing was complete, with the diameter of both the acto- complete absence of a wound per se; for example, large myosin-II ring and the wound decreasing to near zero. membrane-impermeant molecules are incorporated into muscle cell membranes during activity such as exercise Next, the contribution of the cytoskeleton to wound [2]. The emerging picture suggests that healing of wounds healing was evaluated by inhibitors that disrupt actin, in single cells relies on ‘new’ plasma membrane being myosin and the small GTPase Rho, which functions as a supplied by vesicles in a Ca2+-dependent process and that molecular switch to regulate various aspects of actin huge wounds can be sealed effectively using intracellular cytoskeletal function. Both cytochalasin D and latrunculin stores of membranes [3]. It also seems that resealing is a B — inhibitors of actin function — blocked the accumula- process akin to neural transmission in that agents that tion of actin at the periphery of the wound and the ingres- disrupt this transmission also block membrane resealing sion of the wound margin during the healing process. Of after wounding [4]. Because the healing of wounds caused particular interest is the observation that targeting of to single cells is inhibited by agents that disrupt cytoskele- myosin-II to the margin of the wound appeared to be tal structure, such as cytochalasin and nocodazole [5], the independent of actin accumulation at the margin: agents cytoskeleton appears to be necessary for the healing that blocked actin accumulation in response to the injury process. Nevertheless, the mechanism by which did not prevent the accumulation of myosin-II. Wound cytoskeletal function contributes to fusion of the vesicles healing was also aberrant when wounds were induced in with the plasma membrane remains unclear but is an issue the presence of an inhibitory agent that covalently modi- of significant interest. fies Rho and blocks its function.

Groups of cells that form the complex tissues that charac- Similarly, an inhibitor of myosin function, butanedione terize metazoan life also heal themselves ([6,7]; reviewed monoxime (BDM), prevented the normal ingression of the in [8]). Following trauma to skin in fetal vertebrate wound margin, but could only block the recruitment of systems, healing can be so complete as to be essentially myosin-II to the edge of the wound at concentrations scarless, with differentiated structures such as glands and higher than those required to block margin ingression. hair follicles being replaced precisely during the healing Even in the presence of such high concentrations of BDM, process. Studies on the healing of actual epithelial wounds actin appeared to accumulate. Because levels of myosin-II and of wounds made to model epithelia formed from cul- were reduced and not completely absent, it is not yet clear tured cells in vitro show that cells enter the wound site in a whether actin accumulation requires the presence of Dispatch R603

Figure 1

(a) A continuous, cellular purse string forms during wound healing in Xenopus oocytes (a) (b) (c) (i) (after [9]). Interestingly, the red outer ring of actin (arrow) does not superimpose completely on the green, inner ring of myosin (arrow; yellow indicates region of overlap). The dim red ring (double arrow) is the original wound margin before ingression. (This micrograph was kindly provided by Bill Bement). (b,c) A diagram of the actomyosin contractile ring in an echinoderm egg. The (d) (e) (h) double arrow in (b) points to the intact contractile ring. The disrupted ring after treatment with low concentrations of cytochalasin D is shown in (c). Arrows in (c) (f) (g) show the direction in which the ring spreads after cytochalasin disrupts its integrity and indicates tension due to the contractile nature of the ring. (Redrawn from a figure kindly provided by Shinya Inoué and reproduced with Current Biology permission from [17].) (d–g) Cytochalasin- induced breakage and subsequent repair of repair itself and in (g) it is complete (arrow). contributes to morphogenesis during dorsal the cytokinetic contractile ring observed in a (Reproduced with permission from [17].) closure in Drosophila melanogaster (arrows living, Arbacia punctulata egg. Equatorially (h) Posterior midgut invagination in Drosophila point to the actin-rich leading edge). Actin in localized cortical pigment granules mark the melanogaster. Embryo stained with antibody to this embryo is rendered fluorescent via a contractile ring. In (d,e), the pigment granule myosin-II shows four elements (arrows) that chimeric protein comprising green fluorescent band has snapped apart after cytochalasin form a discontinuous, supracellular purse protein and actin-binding protein (D.P.K., treatment (arrows in (d) indicate the broken string (reproduced with permission from [23]). K.A. Edwards, and R.A. Montague, margin of the band). In (f), the band begins to (i) A continuous, supracellular purse string unpublished observations, after [28]).

myosin-II. The high concentration of BDM that is first proposed to drive cytokinesis in animal cells (reviewed required to inhibit myosin function makes this a weak in [13,14]). A cytokinetic contractile ring in an echinoderm aspect of the study. Specifically, at comparable concentra- egg is diagrammed in Figure 1b,c. The idea that such tions BDM has known effects on other cellular targets purse strings were present was made on the basis of several including Ca2+ channels, gap junctions and microtubule- lines of evidence. Distinct forms of the proteins that based motility [10–12]. Particularly disquieting is Bement mediate muscle contraction were found in non-muscle et al.’s observation that both extracellular Ca2+ and micro- cells. Ultrastructural analysis revealed bundles of parallel tubule dynamics are required for the healing process. This and antiparallel actin filaments in the cortex in the cleav- leaves the concern that the target of BDM may be Ca2+ age furrow and immunocytochemical analysis demon- homeostasis or a microtubule-based process, rather than strated myosin-II localization in the furrow. Also, actomyosin function itself. Despite the vagaries of BDM cytokinesis was found to be sensitive to drugs that disrupt specificity and its use, on balance the data on actin and actin function and to microinjection of antibodies against Rho provide compelling evidence for a role for actin-based myosin-II. Molecular genetic and genetic analysis of contractility in wound healing in this model system. cytokinesis in Dictyostelium and the fruit fly also pointed to the involvement of purse strings in cytokinesis. The Finally, microtubules were shown to both positively and underlying assumption is that a contractile function, similar negatively modulate ingression of the wound margin to that observed in the muscle sarcomeres, could be during healing. When applied before wounding, drugs ascribed to such rings even if these rings lack the almost that either disrupt or stabilize microtubules — nocodazole crystalline array of thick and thin filaments that character- and taxol, respectively — inhibit both the healing process ize muscle structure. Elegant micromanipulation experi- and the recruitment of myosin-II to the wound margin. In ments demonstrated that the cellular contractile ring that contrast, when applied after wounding and after the initial drives cytokinesis can indeed exert force and deflect a flex- recruitment of actin and myosin-II, nocodazole actually ible glass needle inserted into a dividing cell [15,16]. enhanced the wound healing process. Of particular interest are a number of observations that The work from Bement et al. [9] reminds us of the extra- such intracellular purse strings could maintain tension in ordinary significance of contractile purse strings in biologi- local regions, even if parts of the purse string were obliter- cal systems (Figure 1). Actomyosin-II purse strings were ated. One elegant demonstration of this was Inoué’s R604 Current Biology, Vol 9 No 16

observation [17] that low concentrations of cytochalasin D Continued analysis of the mechanisms of cytokinesis, caused the furrows in dividing sea urchin eggs to fail, but morphogenesis through the function of supracellular purse only locally, allowing most of the circumference of the strings and wound healing are likely to contribute to an dividing egg to remain constricted (Figure 1b–g). Mechan- overall understanding of the function of purse strings in ical perturbation of the cortex in local regions of furrowing biological systems. A number of key questions remain. For cells supports this notion. These observations illustrate example, what triggers assembly of the actomyosin ring? that, strictly speaking, a purse string in the conventional What are the protein players that are required for the sense of ‘a drawstring for closing certain purses’ provides a assembly, structural integrity and contractile properties of fundamentally inadequate or even misleading model for the ring? How do they function at the molecular level? the function of actin and myosin in the cellular contractile How is their function coordinated to drive function at the ring. Inherent in the design of the cell, but quite unneces- supramolecular, cellular or even supracellular level? sary (and even complicating!) in the design of a purse, is Overall, what is the supramolecular structure of the ring in the periodic attachment of the drawstring to the structure molecular detail and how is the ring disassembled after it that it is designed to close. Thus, the contractile ring is functions? Although the rings that contribute to cytokine- attached to the cortex and/or the overlying plasma mem- sis, development and wound healing all have features in brane at regular intervals with high spatial frequency. common — each ring has actin and myosin as major struc- Despite the inadequacy of the term, the overall concept of tural components — it is likely that some features are a purse string is still useful and powerful, particularly going to be unique to each process. For example, the when the caveat of attachment is remembered as an inte- mechanism that triggers assembly is presumably different gral part of cellular and supracellular purse strings. and must be coordinated with different processes in each case: in the case of cytokinesis, the cell cycle; in morpho- Supracellular purse strings of actomyosin-II were clearly genesis, a particular developmental process; or in healing, revealed by studies on tissue wound healing and on dorsal the infliction of a wound. closure, the morphogenic process whereby the lateral epi- dermis closes up and over the amnioserosa in Drosophila Relevant to the findings of Bement et al. [9] regarding the embryogenesis [6,7,18] (Figure 1i). Subsequently, analysis recruitment of myosin-II to the margin of Xenopus oocyte of various other processes — ventral closure in worms [19], wounds in the absence of actin is the recent finding that the ingression of the centripetal cells during Drosophila myosin-II can form and maintain a ring independently of oogenesis [20], and resistance to the passage of ions and filamentous actin during cell division in budding yeast larger solutes between cells in epithelial sheets (so-called [26]. Such observations provide constraints for the molec- paracellular pathway) [21] — suggested that contractile ular mechanism of purse-string formation in the two purse strings were commonplace in both development and systems. Moreover, the observation that the ring involved tissue homeostasis. Moreover, the observation of continu- in wound healing can be divided into regions that are rich ous, supracellular purse strings suggested that partial purse in actin, rich in myosin and rich in both proteins may have strings might also contribute to the invagination of epithe- important ramifications in terms of the assembly, structure lial cell sheets during morphogenesis through the selective and function of purse strings in general. Recently, muta- apical constriction or ‘wedging’ of cells in the sheet tional analysis in a wide array of model systems has identi- (Figure 1h). Such processes have been shown to have asso- fied a variety of molecular players that contribute to ciated thin filaments [22] and myosin-II [23], and to be sen- cytokinesis and might also contribute to morphogenesis or sitive to drugs that disrupt filamentous actin function [24]. wound healing (reviewed in [14]). For example, in Drosophila, anillin and septins are required for cytokinesis It is essential to point out that, in contrast to cytokinesis, and both proteins colocalize with actin and myosin-II in which the ability of the purse string to generate force during cytokinesis. In contrast, septins, but not anillin, are has been demonstrated, only recently have the purse colocalized with actin and myosin-II in the leading edge of strings that drive morphogenesis been shown to be con- the lateral epidermis during dorsal closure. To date, the tractile. Specifically, the purse string associated with contribution of these proteins during normal morphogene- dorsal closure was shown to be under tension: laser cuts sis or during cellular or tissue responses to wounding has made in the leading edge of the lateral epidermis during not been analyzed. dorsal closure caused cells adjacent to the cut to recoil as if they contained a contractile purse string ([25] and D.P.K., Studies of the mechanism by which purse strings drive the C. Galbraith, K.A. Edwards, M.P.Sheetz and R.A. Mon- cell shape changes that contribute to cell division and tague, unpublished observations). The ingression of morphogenesis have been ongoing for 30 years. Although wound margins during healing suggested that the purse myosin-II is clearly important in these almost-ubiquitous strings associated with wounds are contractile, but a formal processes, it may not be the only motor protein to con- analysis of the mechanical characteristics of the purse tribute to purse string function. Recently, a contractile strings themselves has not yet been performed. purse string has been implicated in phagocytosis [27]. A Dispatch R605

number of different myosins may participate in the overall 19. Williams-Masson EM, Malik AN, Hardin J: An actin-mediated two-step mechanism is required for ventral enclosure of the process of phagocytosis, but only myosin I appears to be C. elegans hypodermis. Development 1997, 124:2889-2901. concentrated in a fashion such that it might contribute to 20. Edwards KA, Kiehart DP: Drosophila nonmuscle myosin II has phagosome closure, through a purse-string-like mecha- multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 1996, 122:1499-1511. nism. As is the case for cytokinesis, morphogenesis and 21. Madara JL: Regulation of the movement of solutes across tight wound healing, a thorough understanding of how purse junctions. Annu Rev Physiol 1998, 60:143-159. strings function in phagocytosis will require a complete 22. Burnside B: Microtubules and microfilaments in newt neurulation. Dev Biol 1971, 26:416-441. list of the molecular players that are required for this 23. Young PE, Pesacreta TC, Kiehart DP: Dynamic changes in the process and a thorough understanding of how they interact distribution of cytoplasmic myosin during Drosophila st embryogenesis. Development 1991, 111:1-14. at molecular resolution. The dawn of the 21 century 24. Wessells NK, Spooner BS, Ash JF, Bradley MO, Luduena MA, Taylor brings a host of new tools and approaches to study these EL, Wrenn JT, Yamada, K: Microfilaments in cellular and important biological structures and promises a new level developmental processes. Science 1971, 171:135-143. 25. Kiehart DP, Galbraith C, Montague RA: Forces for dorsal closure in of understanding of how they function. Drosophila are both parallel and perpendicular to the morphogenic movements that cause the lateral epidermis to spread over the amnioserosa. Mol Biol Cell 1998, 9:146a (abstract). Acknowledgements 26. 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