How Does A Cell Membrane Repair Itself

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COMMENTARY | 01 March 2002

Repairing a torn cell surface: make fashion, lysosomes to the rescue

Paul L. McNeil

Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30904,United states

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Paul L. McNeil

Department of Cellular Biology and Anatomy, Establish of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30904,USA

Online Issn: 1477-9137

Impress Issn: 0021-9533

2002

J Cell Sci (2002) 115 (five): 873–879.

Biological membranes are oftentimes described as `self-sealing' structures. If indeed membranes do have an inherent capacity for repair, does this explicate how a prison cell tin rapidly reseal a very large (1-1000 μmⁱⁱ)disruption in its plasma membrane? It is becoming increasingly clear that, in nucleated fauna cells, the cytoplasm plays an active and essential function in resealing. A rapid and apparently chaotic membrane fusion response is initiated locally in the cytoplasm past the Ca²⁺ that floods in through a disruption: cytoplasmic vesicles are thereby joined with one another(homotypically) and with the surrounding plasma membrane (exocytotically). Every bit a consequence, internal membrane is added to jail cell surface membrane at the disruption site. In the case of big disruptions, this addition is hypothesized to function as a `patch'. In sea urchin eggs, the internal compartment used is the yolk granule. Several recent studies have significantly advanced our understanding of how cells survive disruption-inducing injuries. In fibroblasts, the lysosome has been identified as a key organelle in resealing. Protein markers of the lysosome membrane appear on the surface of fibroblasts at sites of disruption. Antibodies confronting lysosome-specific proteins, introduced into the living fibroblast,inhibit its resealing response. In gastric eptithelial cells, local depolymerization of filamentous actin has been identified every bit a crucial step in resealing: it may function to remove a barrier to lysosome-plasma membrane contact leading to exocytotic fusion. Plasma membrane disruption in epithelial cells induces depolymerization of cortical filamentous actin and, if this depolymerization response is inhibited, resealing is blocked. In the Xenopus egg, the cortical cytoskeleton has been identified as an active participant in mail-resealing repair of disruption-related damage to underlying cell cortex. A striking, highly localized actin polymerization response is appreciable around the margin of cortical defects. A myosin powered contraction occurring inside this newly formed zone of F-actin then drives closure of the defect in a purse-string way.

Introduction

Since the advent of microsurgical techniques in the early on part of the last century, it has been known that fauna cells can survive the experimental creation of very large holes (>1000 μm²) in their surface(reviewed in Chambers and Chambers,1961; Heilbrunn,1956). Remarkably, these initial observations of a truly remarkable cell chapters failed, for many subsequent decades, to generate further interest. This neglect had two causes. First, with the widespread use of microinjection and other methods for breaching the prison cell surface, resealing came to be associated primarily with the laboratory setting rather than with whatsoever natural, biological ane. Resealing permitted cells to survive a microneedle puncture and was therefore a very useful cell holding. It was,however, apparently of picayune biological interest, considering a disruption, it was obvious, was merely a laboratory-generated artifact. Considerable work,which is briefly reviewed here, has clearly demonstrated that the mammalian body, in common with other machines that have moving parts, is not immune under normal operating atmospheric condition from mechanically induced wear and tear. Plasma membrane disruption is a common and normal issue in many mechanically active mammalian tissues, and so resealing, because information technology permits cells to survive this injury, is a response that has central biological significance.

In addition to suffering from a kind of `guilt by association', resealing languished for a second reason. It was thought to have a simple and fiddling explanation. Thus, one time it was established that the main cell surface bulwark torn past the microneedle was a fluid lipid bilayer, resealing became explicable every bit simply the thermodynamically adamant outcome of the well established principle that `Membranes hate edges'(Parsegian et al., 1984). Co-ordinate to this view, even so current in textbooks, resealing is an power inherent in all membranes, a response requiring naught more than than that the torn membrane to exist held at a temperature above its liquid-crystalline transition indicate. This view is no longer tenable, at least in the case of large (>1 μm) disruptions occurring in nucleated beast cells. Rather,every bit outlined here, resealing is now viewed as the outcome of a dynamic and complex mechanism, ane that relies heavily on the participation of numerous cytoplasmic constituents. Contempo work, discussed below, strongly implicates lysosomes and the actin-based cytoskeleton as 2 primal cytoplasmic players in the resealing response and actin/myosin-based contraction in the subsequent repair of wound-associated damage to the cell cortex.

The significance of resealing

Survival is the obvious benefit to a gratis-living jail cell of rapidly resealing a plasma membrane disruption. An open up disruption allows potential toxins, such as Ca²⁺, to flood into the cytosol of the `wounded' jail cell and diffusible cytosolic components, such every bit proteins and ATP, to escape. Rapid resealing prevents a rapid death for the wounded cell.

For long-lived, multicellular organisms, too, resealing may often exist a beneficial and therefore an evolutionarily favored prison cell response. For example,physical insults (accidents or attacks) that disrupt tissue integrity are an obvious cause of plasma membrane disruption. Yet, for a single, isolated tissue injury, it could be argued that resealing is of little result:`one time only' cell replacement costs might be of trivial overall importance, at to the lowest degree in long-lived organisms. Information technology is only in cases in which the cells experiencing a disruption during an injury are both irreplaceable and essential to the continued functioning of the organism that resealing has an indisputable value. Chiefly, this status is fulfilled in the example of,for example, a severed nervus. The private neurons suffering disruptions during the injurious (severing) event cannot be replaced. Therefore they must reseal if they are to survive and subsequently `grow' back to their targets in a successful re-innervation try.

If under normal, physiological circumstances plasma membrane disruption affects a large proportion of a tissue's cell population (e.g. is a frequent event) and/or if it affects large jail cell types, and/or affects cells that are irreplaceable, then resealing might be important under normal every bit well as pathological conditions. Either it reduces the price to the organism of complete prison cell replacement or information technology prevents an accumulating loss of cells essential for the operation of the organism. `Cell wounding', divers as a survivable plasma membrane disruption consequence marked by the uptake into the cytosol of a normally membrane impermeant tracer, is observable under physiological conditions in the endothelium lining the aorta, the epithelium lining the gastrointestinal tract, the epithelium of skin, and the myocytes of cardiac and skeletal musculus (McNeil,1993). The frequent occurrence of disruptions in cardiac(irreplaceable) and skeletal (large) myoctes(Clarke et al., 1995;McNeil and Khakee, 1992),under physiological weather condition, argues that resealing is a fundamental biological response in mammals. It is possible that resealing is also a cost-effective cell adaptation in the several other tissues mentioned.

The proportion of cells classifiable as wounded typically increases as a function of mechanical load. For example, it rises from ∼4% of the myocytes of the triceps muscle of the mouse or rat kept in its muzzle to∼20% after these rodents are exercised past running downhill(Clarke et al., 1993;McNeil and Khakee, 1992),which results in eccentric (high-force-producing) contractions of this muscle. Resealing is therefore an essential, if widely overlooked, office of the many and diverse cells that reside in mechanically challenging tissue environments of the normally functioning mammalian body.

Rapid resealign involves a Ca²⁺-dependent, exocytotic response in nucleated fauna cells

The early studies of Chambers and Heilbrunn established the remarkable chapters animate being cells have for repairing cell surface tears and showed that this chapters is absolutely dependent on the extracellular presence at virtually physiological levels of Ca²⁺(Chambers and Chambers, 1961;Heilbrunn, 1956). For example,they observed that, in the presence of extracellular Ca²⁺, the echinoderm egg exhibits a vigorous cytoplasmic reaction beneath a site of surface trigger-happy and that associated with this response is a minimal loss of intracellular material and the retention of cell viability. In the absenteeism of extracellular Ca²⁺, they observed a rapid elimination out of cytoplasm from the egg through the surface injury site. Heilbrunn termed this Ca²⁺-dependent response, which he and others observed in a broad variety of jail cell types, the `surface precipitation reaction'(Heilbrunn, 1930). Working before information technology was known that the main surface improvidence barrier is a phospholipid bilayer, he hypothesized that exposure of cytoplasm to Ca²⁺ results in the precipitation of a reparative protein barrier over the surface defect.

The first clue to how exactly Ca²⁺ promotes resealing or restoration of disrupted lipid bilayer continuity came from work by Steinhardt et al. (Steinhardt et al.,1994). They found that resealing is inhibited if fibroblasts or sea urchin eggs are showtime injected with botulinum or tetanus toxins and so∼sixty minutes later on wounded by a second microneedle impalement. These toxins are proteases that are thought to specifically target and thereby inactivate members of the SNARE family of proteins required for sure exocytotic events, such equally those occurring at the synapse during neurotransmitter release(Schiavo et al., 1992). Therefore the toxin microinjection experiment suggested that exocytosis is required for resealing. Subsequent work, using both endothelial cells(Miyake and McNeil, 1995) and sea urchin eggs (Bi et al.,1995), confirmed that an exocytotic reaction is rapidly evoked in a Ca²⁺-dependent way by plasma membrane disruption, that this response is localized to the disruption site and that information technology is quantitatively related to disruption magnitude.

What is the function in resealing of this exocytotic delivery of internal membrane to the surface of the wounded prison cell? The plasma membrane adheres to the underlying cortical cytoskeleton (principally filamentous actin). This generates a `membrane tension' (Raucher and Sheetz, 1999) that opposes the `line tension' generated by lipid disordering at the gratis edge of a disruption (reviewed inChernomordik et al., 1987). It is this line tension that, theoretically, promotes lipid period over a disruption site, likewise as the bilayer fusion event required in completing resealing. If therefore the exocytotic events induced past a disruption could somehow reduce membrane tension, the predicted result would be the promotion of resealing through enhanced, line-tension-driven lipid flow. Consistent with this hypothesis is the observation that a rapid (second timescale),Ca²⁺-dependent reduction in membrane tension is induced by membrane disruption (Togo et al., 1999;Togo et al., 2000). Moreover,handling of cells with surface active agents, which might reduce membrane or`surface' tension, enhances resealing and survival(Clarke and McNeil, 1994;Togo et al., 1999).

Vesicle-vesicle fusion

The role for exocytosis that was just described — flow promotion— is hypothesized to be applicative to relatively minor (<i μm diameter) disruptions. The evidence supporting a second hypothesized role for exocytosis — the placement of a reparative `patch' of internally derived membrane over the disruption site — volition now be described.

Electron micrographs of the cortical cytoplasm bordering on endothelial cell disruption sites propose that, in addition to vesicle-plasma membrane fusion (exocytotic fusion), vesicle-vesicle fusion is induced locally by a disruption. The cortical cytoplasm surrounding a disruption displays within seconds afterward its germination a remarkable abundance of abnormally enlarged vesicles (Miyake and McNeil,1995). What is the office of these enlarged vesicles? An reply to this question was suggested by experiments in which sea water was injected along with fluorescent tracers into the cytoplasm of starfish and sea urchin eggs (Terasaki et al., 1997). Fluorescent seawater containing a normal level of Ca²⁺, but non seawater without Ca²⁺, was immediately sequestered as it left the microneedle orifice behind an impermeant barrier. That this barrier was a membrane was confirmed by electron microscopy, staining with lipidic dyes and its measured impermeability not only to small fluorescent dies such as fluorescein stachyose (∼1000 MW) but also to Ca^two+ and fifty-fifty H⁺ ions.

The sea water injection experiment revealed a fundamental concept: cytoplasm by itself, in the absenteeism of plasma membrane and therefore exocytotic events, can form a membrane barrier to prevent farther incursion of the toxic extracellular environment. Moreover, it provided a mechanism consequent with the before electron microscope observations: given the scale (vesicles >10μm in bore form at pipette tips) and rapidity (second or sub second fourth dimension calibration) of the formation of membrane bulwark, the process had to be based on a vesicle-vesicle fusion reaction.

The patch hypothesis

If the process of de novo barrier germination through vesicle-vesicle fusion,just described, occurred in the cytoplasm below a disruption site, and if this barrier was then added by exocytosis to the plasma membrane surrounding the disruption, and then resealing would exist accomplished by a kind of patching mechanism. This `patch hypothesis' tin be stated in more than particular as follows(Fig. 1). Ca²⁺entering through a disruption is hypothesized to evoke a local, cluttered vesicle-vesicle and vesicle—plasma-membrane fusion response. Consequently, a population of abnormally big vesicles is created below the disruption site, and vesicle-plasma membrane fusion events then link the bilayers of some of these vesicles with the plasma membrane. These membrane fusion events continue until a continuous `patch' of membrane has been erected across the disruption site, blocking further entry of fusion-initiating Ca²⁺ ions.

Fig. 1.

The patch hypothesis. (A) An undisturbed cell. The typical subcortical network of F-actin (red) and underlying lysosomes or yolk granules (blueish) is emphasized. (B) A large disruption of the plasma membrane occurs. Caⁱⁱ⁺ entering through the disruption initiates deopolymerization of the F-actin network and triggers accumulation of vesicles (lysosomes or yolk granules) powered by myosin and kinesin motor proteins. (C) The accumulating vesicles begin to fuse with one another to create big patch vesicles. (D)Continuing vesicle-vesicle fusion creates more and larger patch vesicles,while vesicle-plasma membrane fusions, at present possible attributable to dissolution locally of the F-actin barrier, add this membrane to the prison cell surface. (E) A patch of internal membrane added is thereby added. Resealing is now complete.(F) Post-resealing polymerization of F-actin and its contraction mediated by myosin restores subcortical network continuity.

Four fundamental predictions of this `patch' hypothesis have recently been verified in the ocean urchin egg (McNeil et al.,2000). First, native, pre-disruption surface membrane is not present initially over large, resealed disruptions. The membrane covering the disruption site immediately after resealing must therefore be derived from an internal source, as predicted. Second, stratification of organelles induced by egg centrifugation results in a polarization of resealing function. The distribution or availability of internal membrane is therefore a crucial determinant of resealing capacity, as predicted. Third, abnormally large vesicles are readily detected in the cytoplasm underlying a disruption site,both by light and scanning electron microscopy, and the advent of these is rapid and Ca²⁺ dependent(McNeil and Baker, 2001). Vesicle-vesicle fusion is therefore induced locally past Ca²⁺ influx through a disruption, every bit predicted. Fourth, an egg organelle (the yolk granule) displays cytosol-independent homotypic fusion in vitro that is initiated Ca²⁺ (∼x μM threshold) with a T_1/2 of seconds and results in the production of very big (>50 μm bore)vesicles. The egg therefore possesses a vesicle population capable of homotypic fusion that can occur rapidly in the absence of the time consuming,cytosol-dependent priming steps of other homotypic fusion reactions and that can erect large membrane boundaries, as predicted.

The patch hypothesis can explain how extremely large disruptions, requiring substantial membrane replacement, are resealed; how, in fact, some cells are able rapidly to replace their entire surface membrane(Rappaport, 1976). Recent work, discussed below, at present allows the states to name the vesicle population used for patch formation by mammalian cells — lysosomes — and adds, for these cells, an additional early footstep in the mechanism — actin depolymerization. Moreover, information technology is becoming articulate that a wounded cell's repair work continues afterwards information technology has patched the surface bilayer aperture.

What vesicles are used for resealing?

Yolk granules conspicuously are required for resealing of large disruptions fabricated in ocean urchin eggs. In the centrifuge-stratified egg, only the finish containing yolk granules retains resealing competence; the other end, containing endoplasmic reticulum besides as `docked' cortical granules, cannot reseal shear-induced disruptions (both ends probably harbor mitochondria)(McNeil et al., 2000). Moreover, as mentioned above, the yolk granule displays Ca²⁺-initiated fusion in vitro, which has properties expected of a fusion reaction capable of supporting resealing (eastward.g. the speed and capacity to create large boundaries) (McNeil et al., 2000). No other egg fraction similarly displays this Ca²⁺-triggered fusion, except the cortical granule fraction. The cortical granule is, nevertheless, unable to support resealing in the stratified egg, and moreover information technology is absent from resealing-competent fertilized eggs.

What is the organelle used in cells that lack yolk granules? Considerable indirect bear witness had accumulated that pointed to the lysosome. Yolk granules of sea urchin eggs are an acidic compartment(McNeil and Terasaki, 2001)that contains hydrolytic enzymes (Armant et al., 1986), and yolk granules are known to have, in the species studied (Raikhel, 1987;Wallace et al., 1983), an endocytotic origin. Lysosomes of cultured mammalian cells tin be induced past elevated Ca²⁺ levels, both in vitro(Mayorga et al., 1994) and in situ (Bakker et al., 1997), to fuse with one another (homotypically), every bit is required for patch formation. Moreover, Ca²⁺ induces lysosomes to fuse exocytotically with the plasma membrane (Rodriguez et al.,1997), which is another fusion event induced by disruption. This Ca²⁺-regulated lysosomal exocytosis depends on synaptotagmin(Syt)Vii, a lysosomal membrane protein and a member of a Ca²⁺-bounden family of proteins long idea to play a role in fusion mayhap as Ca^two+ sensors(Geppert and Sudhof, 1998). Antibodies against the C2A domain of this protein and recombinant Syt Vii C2A domain peptide both inhibit Ca²⁺-induced (streptolysin-O permebilized) lysosomal exocytosis(Martinez et al., 2000). Antibodies raised against the C2A domain of a squid synaptogamin inhibited resealing in the behemothic squid axon and in cultured PC12 cells(Detrait et al., 2000a;Detrait et al., 2000b). Withal, these axon-resealing studies did not reveal what organelle this antibiotic targeted. Lastly, earlier studies employing fluorescent dyes taken up endocytotically revealed the involvement of the various compartments,including lysosomes, thus labeled in a disruption-induced exocytotic response(Miyake and McNeil, 1995). Once again the organelle involved could not be defined, since this method did not discriminate between the several compartments labeled, for example, endosomes(early on, late) and lysosomes.

Thus it was important to ask more than specifically whether exocytosis of lysosomes is triggered by a plasma membrane disruption. The approach Reddy et al. (Reddy et al., 2001) took in answering this question — to look for the disruption-induced appearance on the cell surface of a lysosmal membrane protein — yielded a striking result. Antibodies against the luminal domain of the lysosome-specific poly peptide, Lamp-i (Granger et al., 1990), practice not stain the surface of undisturbed cells, but strongly stain the surface of wounded cells(Fig. 2). This surface exposure of Lamp-1 is Caⁱⁱ⁺-dependent and localized to disruption sites made with a microneedle. To test the functional importance in resealing of the disruption-induced lysosomal exocytosis, Reddy et al. introduced into living cells antibodies to the C2A (calcium-binding) domain of Syt VII, as well equally recombinant peptide fragments of the whole protein. Both of these reagents inhibited the surface advent of the Lamp-1 luminal domain and jail cell resealing. These inhibitory effects were observed when the disruption result being monitored was too the route of access of reagent to cytoplasm. Inactivation must therefore have been extremely rapid, since resealing is generally complete in <ninety sec in these cells. In an independent examination of the function of lysosomes, antibodies against the cytosolic domain of Lamp-ane, which accept a lysosome-aggregating activity, also inhibited fusion.

Fig. 2.

Staining of a resealed fibroblast with antibodies against a luminal domain of the lyososome-specific poly peptide, Lamp-1. The correct mitt side of this cell was severed when the civilization dish it was growing in was scratched with a needle (the carmine streak on the right indicates the needle path). Staining with anti-Lamp-1 was then performed on the living, resealed cell, limiting detection to surface exposed antigen but. Surface exposure of Lamp-1,indicated by the staining, is evident over the resealed disruption site. This provides stiff testify that the local exocytotic response evoked by a disruption utilizes lysosomes. (Photomicrograph courtesy of Norma Andrews,Yale University.)

These studies, it must be pointed out, practise non rule out the participation of other organelles in resealing. When the cortical granules of the bounding main urchin are `undocked' by treatments with stachyose, resealing is reversibly inhibited(Bi et al., 1995). This is indirect evidence for a cortical granule contribution to resealing, although it remains unclear how specific the stachyose treatment is for cortical granules. Moreover, ii separate pools of vesicles, identified on the ground of the timing of their exocytosis and on their susceptibility to myosin/kinesin inhibitors, are required for resealing in urchin eggs(Bi et al., 1997). This is indirect testify that, in the sea urchin egg, resealing might use both yolk granules and cortical granules, as well every bit other, every bit-withal-unidentified organelles. It besides remains possible that, in other `specialized' prison cell types,organelle compartments other than, or in addition to, lysosomes are mobilized for resealing.

Rescue by lysosomes: a dangerous expedient?

As mentioned above, plasma membrane disruption is a mutual and normal jail cell injury in vivo. Therefore, the lysosomal exocytotic response required for resealing must occur constitutively and indeed at an enhanced rate during, for case, certain forms of practise. What prevents consequent release of a bombardment of potentially destructive hydrolytic enzymes from becoming a net disadvantage to the organism? Two potentially relevant points can be fabricated in answer to this question. First, lysosomal enzymes have a pH optimum of∼3.5. Thus, release into the extracellular environment of a mammalian tissue, which has a pH of ∼7.4, severely curtails their activeness level. Second, many cells, including liver hepatocytes, which are well placed as a filter of claret, possess jail cell surface mannose-vi-phosphate receptors capable of mediating endocytotic uptake of lysosomal enzymes(von Figura and Hasilik,1986). Therefore, lysosomal enzymes released equally a consequence of cell wounding in vivo will display sub-optimal hydrolytic activity and will be apace scavenged from the extracellular environment.

Making way for lysosomes: disruption-induced cortical depolymerization

Regulated secretory vesicles are, in most cells, intimately associated with the plasma membrane prior to receipt of an exocytosis-inducing indicate. Different these `predocked' vesicles, lysosomes are subcortical organelles and therefore a filamentous actin (F-actin) barrier stands in the way of fusion-productive contact between lysosomes and betwixt lysosomes and the plasma membrane. Recent work suggests that Ca^two+-initiated depolymerization of cortical actin is a pre-requisite for resealing(Miyake et al., 2001). Thus,in rat GM1 epithelial cells, an apparent reduction in F-actin, visualized by staining with phaloidin, was observed at disruption sites. Menstruum cytofluorometric analysis of phaloidin staining of populations of wounded cells provided a quantitative confirmation that disruption reduces F-actin levels in cells and showed that the subtract is Ca²⁺-dependent. Drug-induced stabilization, or biologically induced enhancement, of the cortical F-actin cytoskeleton severely decreases resealing. By contrast, actin depolymerization, induced by DNAse 1, enhances resealing. Dissolution of a cortical filamentous actin bulwark standing in the way of lysosome-lysosome and lysosome-plasma membrane contact must therefore be a crucial early on pace in the resealing mechanism (Fig. 1).

Before studies suggested that the cytoskeleton is an help, likewise as an obstacle, to resealing. Antibodies to kinesin and a general inhibitor of myosins, both inhibited resealing (Bi et al., 1997; Steinhardt et al.,1994). Ane possible role for these motors might exist to move vesicles into the disruption site and hence to promote contacts leading to the vesicle-vesicle and exocytotic fusion events required for resealing. The remarkable concentration of vesicles observed by electron microscopy to occupy cortical disruption sites in endothelial cells(Miyake and McNeil, 1995) is consistent with this theorize, but, and then far, the disruption-induced vesicular movements that might be powered by kinesin and/or myosin take not been straight observable.

Continuing repairs and defensive preparations

Resealing prevents disaster (e.g. prison cell expiry), simply restoration of full jail cell function might require additional, follow-upwards repairs. For case, a membrane-disrupting strength penetrating deeply into the cell volition necessarily damage the cortical cytoskeleton as well. Even in the absence of such direct damage, the F-actin depolymerization response consequent to disruption (come across higher up) is predicted to disrupt normal cytoskeletal structure locally. Contempo work has suggested how repair of such damage is accomplished(Bement et al., 1999;Mandato and Bement, 2001). The cortex underlying a light amplification by stimulated emission of radiation wound to a Xenopus oocyte is initially(t=five-x minutes, for a 50 μm diameter wound) depleted of its normal meshwork of F-actin and associated myosin 2. Whether this F-actin depletion(henceforth referred to as the cortical wound) is caused by the light amplification by stimulated emission of radiation injury or by the disruption-induced depolymerization response just described above is not known but remains an interesting question. In any example, repair of the cortical wound is initiated by assembly of an F-actin and myosin two network in a zone bordering the wound site. A narrow ring, consisting of full-bodied F-actin and myosin ii, appears within this assembly zone, and this band is demonstrably contractile (Fig. 3). This contraction, which occurs in a bag-string mode, is required for closure of the cortical wound. The final phase of closure is associated with the germination of `fingers' of F-actin along the sides of the now closely apposed wound borders. Similar actin structures are formed by split up cells contacting each other during dorsal closure in Drosophila, some other of numerous remarkable parallels between tissue(multicellular repair of tissue defects) and private cell (membrane disruption) wound healing (Woolley and Martin, 2000). Apparent contacts between these opposing fingers are observed, and their contraction might ability final closure of the cortical wound.

Fig. three.

(A) Post sealing repair of wound-related damage to the cortical F-actin network. A Xenopus oocyte injected with fluorescent actin was wounded(circular profile) with a laser and and then imaged at intervals of 3 minutes. An intensely labeled zone of F-actin assembly is seen around the wound site in the start console and, in subsequent panels, is seen to contract,purse-string-fashion, restoring continuity to the cortical actin network. (B)Experimental assay of the zone of actin polymerization indicates that it contains at its interface with the wound site a `contractile array' consisting of F-actin and myosin 2. Finger-like, polymerization-dependent protrusions of F-actin extend into the wound site and may facilitate last closure.(Micrographs and drawing courtesy of C. Mandato and Due west. Bement, Academy of Wisconsin.)

Afterwards resealing a disruption, and repairing associated cortical impairment, can a cell so set up itself to better withstand futurity injury? Fibroblasts reseal a 2d plasma membrane disruption more chop-chop than a first made ten minutes before (Togo et al.,1999). Drug inhibitor and activator experiments suggest that this`facilitated resealing' is dependent on enhanced protein kinase C activity and enhanced vesicle production by the Golgi appliance. Such an enhancement of vesicle production might target lysosomes, which are, of course, supplied with membrane (and enzymes) past Golgi-derived shuttle vesicles. An increase in the size and/or number of lysosomes should enhance resealing (see above) and might, therefore, constitute the mechanistic basis of the facilitated resealing response.

Future challenges

A disarming hypothesis for explaining how cells reseal large tears in their surface tin now be outlined, but many questions remain, especially at the molecular level. Caⁱⁱ⁺ inbound through a disruption is the likely trigger of the vesicle-vesicle and vesicle-plasma membrane fusion events that event in the construction of a vesicular patch and its annealing to the disruption margins. Recent work clearly identifies synaptotagmin as a central thespian in the resealing response and suggests that this protein, which is known to accept ii Ca²⁺-binding domains, is an important Ca²⁺-sensing component of the fusion response. This hypothesized Ca²⁺-sensing role, which could greatly strengthen the full general notion that synaptotagmin has a Ca²⁺-sensing part in membrane fusion, obviously warrants further experimentation. And what is the identity of the remaining components of the fusion machinery? Proteins of the SNARE family are implicated in the exocytotic events of resealing, but their involvement in the vesicle-vesicle fusion step of resealing has not been confirmed. Moreover, the in vitro fusion reaction has some unorthodox properties that suggest that its molecular elucidation could yield some surprises: dissimilar other homotypic fusion systems, reconstituted in vitro, yolk granule fusion does non require cytosol, ATP or GTP, and it is, moreover,extremely rapid (on a timescale of seconds rather than many minutes). The molecular basis of the actin depolymerization response also remains unknown. Finally, it is now clear that the response to plasma membrane disruption is a complex and dynamic one. Additional interesting adaptations to disruption surely remain to be discovered. Does, for example, the wounded cell switch on,in addition to the cortical-repair response, other `clean-upward' activities? Are there longer-term changes in wounded jail cell behavior, dependent perhaps on changed patterns of gene expression, that constitute an adaptive response to mechanical stress? Our recently rekindled interest in how cells cope with a life threatening but normal and perhaps, in the life of many cells, inevitable injury promises answers to these and many more unforeseen questions arising in this rapidly emerging field of prison cell biology.

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