Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string

The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply excluded from the periphery by some general mechanism. The results indicate that the actomyosin purse string is not the only mechanism that can mediate wound closure in single cells.     

Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.

BACKGROUND: Both single cells and multicellular systems rapidly heal physical insults but are thought to do so by distinctly different mechanisms. Wounds in single cells heal by calcium-dependent membrane fusion, whereas multicellular wounds heal by a variety of different mechanisms, including circumferential contraction of an actomyosin 'purse string' that assembles around wound borders and is dependent upon the small GTPase Rho. RESULTS: We investigated healing of puncture wounds made in Xenopus oocytes, a single-cell system. Oocyte wounds rapidly assumed a circular morphology and constricted circumferentially, coincident with the recruitment of filamentous actin (F-actin) and myosin-II to the wound borders. Surprisingly, recruitment of myosin-II to wound borders occurred before that of F-actin. Further, experimental disruption of F-actin prevented healing but did not prevent myosin-II recruitment. Actomyosin purse-string assembly and closure was dependent on Rho GTPases and extracellular calcium. Wounding resulted in reorganization of microtubules into an array similar to that which forms during cytokinesis in Xenopus embryos. Experimental perturbation of oocyte microtubules before wounding inhibited actomyosin recruitment and wound closure, whereas depolymerization of microtubules after wounding accelerated wound closure. CONCLUSIONS: We conclude the following: actomyosin purse strings can close single-cell wounds; myosin-II is recruited to wound borders independently of F-actin; purse-string assembly is dependent on a Rho GTPase; and purse-string assembly and closure are controlled by microtubules. More generally, the results indicate that actomyosin purse strings have been co-opted through evolution to dispatch a broad variety of single-cell and multicellular processes, including wound healing, cytokinesis and morphogenesis.     

Wound healing activity of NOE-aspirin: a pre-clinical study.

Aspirin, one of the oldest non-steroidal anti-inflammatory drugs, impedes tissue repair by virtue of retarding inflammation. The present study was undertaken to find out if linking of nitrooxyethyl ester to aspirin reverses its healing-depressant propensity. Nitrooxyethyl ester of aspirin (NOE-Asp) was synthesized in our laboratory through well-established synthetic pathway, starting from aspirin through esterification with ethylene glycol and nitration with a mixture of nitric and sulfuric acids at 0 degrees C. The effect of NOE-Asp on phases of healing such as collagenation, wound contraction and epithelialization and on scar size of healed wound was evaluated in three wound models-incision, dead space, and excision wounds. To assess its influence on the oxidative stress, the levels of glutathione (GSH) and thiobarbiturate reactive species (TBARS) were also determined in 10-day-old granulation tissue. NOE-Asp was further screened for its anti-inflammatory activity in rat paw edema model. NOE-Asp promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction, and epithelialization phases of healing. NOE-Asp also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to aspirin. The results vindicate our assumption that the esterification of aspirin with nirooxyethyl group reverses the healing-suppressant effect of aspirin. The compound also showed equipotent anti-inflammatory activity as aspirin.     

Wound healing in X-irradiated mammalian skin.

Certain parameters of wound healing were investigated in mouse skin given 950 rads and 3325 rads of X-irradiation at various times relative to wounding. Increased inflammation, delayed dermal regeneration, and delayed contraction were noted in all irradiated groups. The activation of surrounding hair follicles, a process that usually accompanies wounding in mouse skin, occurred earlier, over a shorter elapsed time, and over a greater area of skin in animals irradiated prior to wounding than in the controls or in those irradiated and wounded simultaneously. Epidermal mitotic activity in wounds made at the time of irradiation was initially depressed but recovered by the second postoperative day. Wounds in pre-irradiated animals gave an unexpected result. They responded with an immediate burst of mitotic activity without the usual 24-hr lag that was seen in controls. In the pre-irradiated specimens a substantial number of cells appeared to die after dividing.     

Wound healing: GTPases flux their muscles

An article by Houk et al. (2012) in Cell provides insight into the mechanisms confining membrane protrusions to the front of migrating neutrophils. The authors rule out a role for diffusion of inhibitory signals and show that membrane tension is necessary and sufficient to restrict signals that lead to protrusions.     

Wound healing in the trematode Schistosoma

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.     

Wound healing: calcium flashes illuminate early events

What are the earliest signals produced at a wound edge that mobilise epithelial cells to heal the wound? Live analysis of wound healing in the worm Caenorhabditis elegans shows that calcium may be the key early trigger.     

Wound healing in tadpole tailfin pieces in vitro.

Explants of tail fins from R. catesbeiana tadpoles undergo reepithelialization of their cut surfaces (healing) when cultured in vitro in Hanks' balanced salt solution at 22 degrees C. Healing is initiated early and closure of the wound is complete by 12 to 24 hours. Morphogenesis continues for several days as further reorganization and migration of epidermal cells from the regions adjacent to the wound margins take place. The addition of serum to the culture media improves the general appearance of these tissues and promotes healing. The rate of healing is affected by temperature. Tail fins maintained at 10 degrees C do not heal while fins maintained at 30 degrees and 37 degrees, although healing more rapidly than at 22 degrees, undergo progressive degeneration in culture. Epidermal cell movements were also studied in explants consisting of a combination of intact tail fin plus tail fin deprived of its epithelium. Rapid and extensive migration of epidermal cells from the intact tail fin across the collagen lamella of the stripped fin is observed.     

Wound healing profile of topical xylocaine preparations in rodents.

Local anesthetics are commonly used for alleviating pain in many clinical conditions associated with wound. Present study is undertaken to unravel the implications of two xylocaine preparations on dermal wound repair in albino rats. Application of xylocaine either as jelly or ointment significantly reduced skin wound breaking strength and promoted re-epithelization. While bases of xylocaine jelly and ointment had no action on skin wound breaking strength but enhanced re-epithelization. Current findings, suggest that prior knowledge of the effects of substances incorporated in topical preparations on wound repair is important for their judicious use and selection of preparation in relevant clinical conditions.     

Wound healing in the cornea of the chick embryo

Spatio-temporal changes in the shapes of the epithelial cells in culture were followed with the aid of scanning electron microscopy. On a substratum that enables the epithelium to spread extensively, the first remarkable change in shapes of the cells occurred at the margin of epithelium at 12 h of culture. The marginal cells formed leading edges with filo- or lamellipodia, flattened, and lost microvilli on surface. In accordance with those changes, the borderlines among cells became almost indiscrenible. Flattening of the cells was the essential characteristic associated with active epithelial spreading throughout the culture period. Elongation of cells of intermediate zone at right angles to the direction of the locomotion of the marginal cells at 24 h of culture was the second significant change. As the third, the change from the ordinary pentagonal or hexagonal to extraordinary tetragonal or other polygonal shapes, with or without irregular margins, began in cells of the intermediate area at 24 h and propagated to those in inner area. The active deformation of the inner cells with no space in which to move was considered to play some role in the extensive epithelial spreading.     

Wound healing in the primitive deep layer of the young chick blastoderm.

Wound healing in the primitive deep layer of stage 4 chick blastoderms was studied in vitro by cinemicrophotography of living cultures and by photomicroscopy, scanning- and transmission electron microscopy after fixation. Experimental wounds with an average diameter of 0.3 mm healed completely within 2 to 4 h through migration of the cells at their rims. Healing occurred in mesenchyme-free areas, providing us with a purely epithelial reaction. The rim cells of the primitive deep layer formed extensions at their free flank, described as fila, filopodia, lamellae and lamellipodia. They were already present in blastoderms fixed at the earliest after the intervention. This reaction was ascribed to the elimination of a normal fellow cell at the side of the rim cell facing the defect. Movement of the rim cell ceased upon meeting another cell with the same polarity. At this moment lamellipodia disappeared as suddenly as they had formed, and the number of fila and filopodia decreased. We believe that the chick blastoderm's primitive deep layer might be appropriate for analysis of the factors governing primary epithelial wound healing.     

Wound healing in the imaginal discs of Drosophila. I. Scanning electron microscopy of normal and healing wing discs.

Scanning electron microscopy was used to investigate the morphology of intact imaginal wing discs of third-instar larvae of Drosophila melanogaster. The disc stalk, nerve and tracheal entries and the surface ultrastructure of the columnar cells, the peripodial membrane cells, and the adepithelial cells are described. The behavior of various fragments of the wing disc during culture in vivo was also studied. After injuring a wing disc by cuts with a tungsten needle, during the first day of culture the epithelium curls and the wound surface contracts. Subsequent closure of the wound in $\text{3}\text{4}$ and $\text{1}\text{4}$ sectors, in fragments generated by straight cuts and in central squares, leads to the confrontations of cells from formerly separate positions, as was proposed in connection with the polar coordinate model of French, Bryant, and Bryant [(1976). Pattern regulation in epimorphic fields. Science193, 969–981]. Wound healing comprises three steps: (1) Cell debris is removed; (2) occasional cell processes span the wound; (3) all cells at the wound edge contact cells on the opposite side. After 2–3 days, a continuous epithelium is re-established. The tissue distortion may lead to transient contacts of the columnar epithelium with the peripodial membrane and with itself. The latter can explain the occasional duplications of structures which, according to the fate map, arise from near the wound edge, and which have been previously reported from cultured imaginal disc fragments. The tissue movements appear to be due to the contractile properties of individual cells.     

Wound healing in the liver with particular reference to stem cells.

The efficiency of liver regeneration in response to the loss of hepatocytes is widely acknowledged, and this is usually accomplished by the triggering of normally proliferatively quiescent hepatocytes into the cell cycle. However, when regeneration is defective, tortuous ductular structures, initially continuous with the biliary tree, proliferate and migrate into the surrounding hepatocyte parenchyma. In humans, these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals, similar ductal cells are usually called oval cells, and their association with impaired regeneration has led to the conclusion that they are the progeny of facultative stem cells. Oval cells are of considerable biological interest as they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. This review proposes that the liver harbours stem cells that are located in the biliary epithelium, that oval cells are the progeny of these stem cells, and that these cells can undergo massive expansion in their numbers before differentiating into hepatocytes. This is a conditional process that only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical, continually renewing, stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the ability to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.     

The transformative power of story for healing

One of our goals in this session was, not just to talk about the healing power of narrative, but to experience it as well. Louise Profeit-LeBlanc is one of the presenters we invited specifically because of her skills as a storyteller. She has been heavily involved for several years as both an organizer and a participant in the Yukon Storytelling Festival, held every year in late May in Whitehorse. Woven into her presentation is a useful framework for differentiating various kinds of stories. As she tells us a series of stories, she takes us through a wide range of emotions from grief and loss to laughter and awe. For each of her stories, she gives us some personal contextual information that adds to the story’s meaning and helps us appreciate its significance. Her final story, in particular, is the kind of traditional story that has probably existed for a very long time. Such stories may be told with slightly different emphases, depending on the occasion, but they carry wisdom and value for every generation that hears them.