Plasma Membrane Repair: A Central Process for Maintaining Cellular Homeostasis

Abstract

Plasma membrane repair is a conserved cellular response mediating active resealing of membrane disruptions to maintain homeostasis and prevent cell death and progression of multiple diseases. Cell membrane repair repurposes mechanisms from various cellular functions, including vesicle trafficking, exocytosis, and endocytosis, to mend the broken membrane. Recent studies increased our understanding of membrane repair by establishing the molecular machinery contributing to membrane resealing. Here, we review some of the key proteins linked to cell membrane repair.

A eukaryotic cell is separated from the extracellular environment by a plasma membrane composed of a phospholipid bilayer containing proteins that regulate transit of molecules into and out of the cell. Loss of this barrier function can lead to compromised cellular homeostasis and death of the cell. Most cells are subjected to mechanical or chemical stresses that can disrupt the plasma membrane; thus there is strong selective pressure to ensure the integrity of this membrane. The inherent nature of phospholipids and early work with lysosomes indicated that the plasma membrane would thermodynamically reseal after disruption (112). While this is true of simple lipid bilayers or small membrane disruptions (FIGURE 1A), the plasma membrane contains integral proteins that interact with the cytoskeleton and extracellular matrix to support numerous cellular functions. These interactions create mechanical tension on the plasma membrane that holds the membrane open after disruption (25, 114). Such disruptions allow intracellular components to escape the cell and potentially permit toxic levels of Ca²⁺, oxidants, and other components of the extracellular milieu to enter the cell. Thus, if these disruptions are not closed rapidly, it may lead to the death of the cell. As a result, cells have evolved active methods to reseal plasma membrane disruptions in which normal cellular responses are repurposed to mend the broken membrane (70, 106, 130) through a process called membrane repair.

FIGURE 1.

Models of the plasma membrane repair process

A: thermodynamic resealing occurs spontaneously due to tension produced by the disordered arrangement of the membrane phospholipids at the open edge of the break. This process is the most likely route of resealing for membrane breaks of ≤1 μm in diameter. B: exocytosis can contribute by trafficking intracellular vesicles to the wounded area where they can fuse with each other and the injured membrane to form a repair patch. C: wound constriction is mediated by caveolae. During this process, caveolae cluster and fuse around larger wounds, leading to wound constriction and intracellular fusion of caveolar endosomes. D: budding/blebbing of the membrane portion containing the wound site with release of the newly formed vesicles into the extracellular space also involves exocytosis. E: exocytosis of an intracellular patch and fusion to the wound site could result in the extracellular release or “shedding” of the wound site. F: endocytosis of wounds occurs via invagination of caveolar vesicles and subsequent intracellular fusion of caveolae.

The idea of facilitated membrane repair was supported by earlier work (23, 59) before the concept was formally presented by McNeil and colleagues, who initially showed that plasma membrane disruptions and repair occur in vivo and that damaged cells reseal by recruitment of intracellular vesicles to form a repair patch in an extracellular Ca²⁺-dependent manner (FIGURE 1B) (12, 96–98, 106, 126). Membrane repair can also involve fusion of vesicles at the injury site or into the proximal plasma membrane. Constriction of the membrane around disruptions can also contribute to membrane repair (FIGURE 1C) (9). Endocytotic mechanisms may be involved in resealing of larger membrane disruptions, whereas smaller disruptions of <100 nm reseal through budding and exocytosis (FIGURE 1D). Repair through budding involves pinching the membrane at the injured site and shedding the injured membrane into the extracellular space (FIGURE 1E) (4, 74, 79). Endocytosis is also thought to contribute to membrane repair by internalization of the injured membrane (FIGURE 1F) (70, 74). How and under what specific conditions these mechanisms contribute to membrane repair is still an area of investigation. These non-exclusive mechanisms could be relevant in a cell-type and context-dependent fashion. It is clear that compromised membrane repair contributes to pathophysiology in a number of different tissues and that it is linked to muscular dystrophy, heart failure, neurodegeneration, and other diseases (5, 7, 24, 27, 50, 72, 133, 140). Despite the importance of membrane repair in cellular function, the field has only recently begun to expand with the discovery of more proteins linked to resealing damaged cell membranes. The focus of this review is to identify several proteins currently linked to membrane repair and to describe some of the key findings on their functions.

Ferlin Family (Dysferlin/Myoferlin/Otoferlin)

Dysferlin is a type II transmembrane protein from the ferlin family that localizes to the plasma membrane and T-tubules of muscle fibers (FIGURE 2) (1, 2). Dysferlin was initially identified as the target gene mutated in Myoshi Myopathy (MM) and limb-girdle muscular dystrophy (LGMD) type 2B (6, 71, 87, 132). Subsequent dysferlin knockout mouse studies showed that muscle fibers (63) and hearts (24, 56) from these animals demonstrated a decreased ability to reseal the plasma membrane (5), leading to muscular dystrophy and late-onset cardiomyopathy (5, 24). These results identified dysferlin as the first protein to contribute directly to membrane repair in striated muscles, resulting in dysferlin's prominent role in the membrane repair literature (26). Subsequent studies linked dysferlin to roles in membrane receptor recycling, endocytosis, vesicle trafficking, membrane turnover, focal adhesion, and ATP-dependent intercellular signaling and modulation of the immune system (31, 34, 36, 47, 58, 109, 139, 141). Recent in vivo studies indicated that dysferlin may function in the maintenance of T-tubule structure (78) and that the principal function of dysferlin may be at the T-tubules rather than in sarcolemmal membrane (77). Additional studies will be necessary to establish the extent that these findings alter the current understanding of dysferlin in membrane repair; however, it is clear that much is still unknown about dysferlin function.

FIGURE 2.

Major membrane repair proteins and their hypothesized roles in the repair process

Dysferlin's interaction with AHNAK is regulated by calpain, and cleavage of dysferlin can result in additional subunits that function in repair. Calpain may also regulate cytoskeletal structure and sarcomere remodeling. AHNAK may aid in cytoskeletal remodeling. MG53/TRIM72 and dysferlin form a vesicle lattice to close the wound. A: affixin, which also binds dysferlin, localizes to focal adhesions and may organize actin. B: ESCRT and acid sphingomyelinase (ASM) facilitate exocytosis and endocytosis. ESCRT has been found to be involved in both endocytosis and budding. ESCRT III can be recruited to the membrane, followed by blebbing of the membrane and shedding of the wound. ASM is secreted and cleaves sphingomyelin to generate ceramide, leading to membrane invagination of the injury site. C: the annexin and S100A10 complex binds dysferlin and may recruit AHNAK to the membrane due to annexin's ability to bind lipid rafts. Annexin/S100A10 may also bridge adjacent phospholipids to form endosomes. Annexin accumulates at the neck of membrane blebs to mediate microvesicle release. Annexin A6 may also “cap” the membrane repair patch. D: synaptotagmin and SNARE proteins interact at the plasma membrane via a conformational change in synaptotagmin present on synaptic vesicles to fuse the vesicles with the membrane.

Other members of the ferlin family also have been linked to membrane repair. For example, myoferlin shares a 56% homology to dysferlin and is expressed during muscle development to facilitate myoblast fusion (41, 139). Wild-type mice show low expression of myoferlin; however, increased expression was observed in mdx and gamma-sarcoglycan-null mouse models of muscular dystrophy (37, 141). Transgenic mouse experiments showed that myoferlin overexpression could compensate for dysferlin in membrane repair; however, myoferlin does not prevent all dystrophy symptoms (90). Another dysferlin family member, otoferlin, regulates synaptic vesicle exocytosis in cochlear hair cells and may be involved in membrane repair specifically within these cells (75, 121).

Calpain

After membrane disruption, rapid reorganization of cytoskeletal elements at the site of injury is necessary. Treatment with the actin-depolymerizing enzyme DNase I significantly enhanced resealing, demonstrating that disassembly of the actin skeleton is important in membrane resealing (107). Calpain 3 is a cysteine protease that cleaves cytoskeletal proteins, such as talin and vimentin, and may aid in early remodeling of various proteins during membrane repair as cleaved fragments of these cytoskeletal proteins could not be recovered in calpain-null cells (101). Calpain 3 cleaves AHNAK, inhibits AHNAK's interactions with dysferlin and myoferlin, and regulates AHNAK protein turnover (FIGURE 2). Through these actions, calpain regulates cytoskeletal structure and interaction of the cytoskeleton with the cell membrane (34, 67). In fact, loss of calpain 3 in particular has been shown to cause limb girdle muscular dystrophy (119). Calpain has been shown to be required for calcium-dependent membrane repair, since knockout of the small subunit of the calpain enzyme results in failure to reseal after laser-induced membrane disruption (101). Calpain could contribute to membrane repair through other mechanisms as well. Studies suggest that calpain can remodel sarcomeres since calpain 3 binds titin and is present in other regions of the sarcomere, such as the Z disk, costameres, and myotendinous junctions (45). Thus the role of calpain may be to increase local loosening of the sarcomere via proteolysis and facilitate removal of damaged and cleaved proteins by the proteosome (8). Other studies suggest a role for calpain 3 in post-membrane repair sarcolemmal remodeling because loss of calpain in limb girdle muscular dystrophy leads to disorganization of myofibers and a lack of organized sarcomeres and sarcomere proteins in myotubes (45, 67, 100). Finally, calpain activity leads to dysferlin cleavage to produce subunits that function in membrane repair (116).

MG53/TRIM72

Mitsugumin 53 (MG53/TRIM72) has been shown to be a vital component of the membrane repair machinery in several cell types (18, 21, 44, 80). It is a member of the tripartite motif family of E3 ubiquitin ligases (TRIM72) that was originally identified in an immunoproteomic library screen for muscle enriched proteins (136). Although native MG53/TRIM72 protein was initially thought to be found only in skeletal and cardiac muscle (18), recent studies have shown expression and membrane repair function in other tissues (44, 73, 80). Along with native protein repair capacity, overexpression of MG53/TRIM72 in cells that do not express MG53/TRIM72 also shows protective effects against membrane injury (137). MG53/TRIM72-null mice display defective membrane repair (18), progressive myopathy (18), and increased susceptibility to injury in the heart (21, 135), lungs (73, 80), and kidneys (44).

MG53/TRIM72 interacts with phosphatidylserine to associate with intracellular vesicles and the inner leaflet of the plasma membrane (18) (FIGURE 3). Once a cell is injured, MG53/TRIM72 is thought to react to the oxidized extracellular environment entering the cell by forming higher molecular weight units (69). MG53/TRIM72-tethered vesicles traffic to the membrane disruption, allowing vesicles to fuse and patch the injured membrane (18). This process appears to involve a dysferlin and Caveolin-3 (Cav-3) containing complex that regulates repair in the sarcolemma (20, 134). A study using ballistic injury and super-resolution microscopy of human myotubes showed that MG53/TRIM72 is recruited quickly to the membrane (2 s), followed by dysferlin ∼10 s post-injury (84). MG53/TRIM72 and dysferlin form a lattice that fills the wound area with vesicles and closes the wound (84).

FIGURE 3.

Proposed roles of MG53/TRIM72 in mediating membrane repair

MG53/TRIM72 interacts with phosphatidylserine in the plasma membrane in a complex containing dysferlin and Cav-3. MG53/TRIM72 and dysferlin close the membrane wound with vesicles. Vesicle transport is facilitated by myosin motor proteins. Cav-3 may regulate MG53/TRIM72-mediated membrane fusion and is enriched in caveolae or plasma membrane invaginations. PTRF may aid in the formation and stabilization of these caveolae by interaction with Cav-3 and MG53/TRIM72 through cholesterol. PTRF also binds and may help localize dysferlin.

Interestingly, when myotubes are injured in media containing exogenous recombinant human MG53/TRIM72 protein (rhMG53), the protein can be seen to localize to the injury site (137). This mechanism is similar to intracellular MG53/TRIM72 binding to exposed phosphatidylserine (18) and does not require endogenous MG53/TRIM72 or dysferlin (137). This association with injury sites can increase membrane resealing (137). Application of rhMG53 to animal models of various diseases, such as muscular dystrophy (137), myocardial infarct (88), lung injury (73, 80), compartment syndrome (28), and acute kidney injury (44) can reduce the pathology in these models. While this property of the protein is dependent on the phosphatidylserine binding capacity of rhMG53, it is possible that other aspects of the protein function, such as regulation of intracellular signaling, could also contribute to this ability to increase membrane repair capacity (55, 76, 83, 88). Further studies should determine the rhMG53 extracellular mechanism of action and assess its potential as a therapeutic agent.

Caveolin

Caveolin family (Cav-1, -2, and -3) proteins are 21- to 24-kDa integral membrane proteins enriched in invaginations of the plasma membrane (caveolae) and are involved in membrane transport (82) (FIGURES 1C AND 3). Cav-3 is muscle specific and has been most closely linked with membrane repair. Mutations in Cav-3 cause autosomal-dominant LGMD1C and autosomal-dominant rippling muscle disease (AD-RMD) (110). Cav-3 has been shown by co-immunoprecipitation to interact with MG53/TRIM72 and dysferlin (20), and Cav-3 overexpression regulates membrane fusion events by downregulating MG53/TRIM72-induced membrane budding and preventing development of filopodia-like structures (19). Disruption of the Cav-3/dysferlin/MG53 complex can affect the localization and membrane repair function of the other components. For example, dominant negative Cav-3 mutations associated with the development of muscular dystrophy have been shown to cause retention of MG53/TRIM72 (20) or dysferlin (61) in the Golgi apparatus and loss of membrane repair capacity. However, other studies using ultrastructural analysis of dysferlin trafficking showed that dysferlin can still reach the plasma membrane in the absence of Cav-3 but that it is rapidly endocytosed (60).

Polymerase-1 and Transcriptase Release Factor (PTRF/Cavin-1/Cav-p60)

PTRF may aid in the formation of caveolae at the plasma membrane since PTRF localizes to caveolae, expression of PTRF is sufficient for caveolae formation, and loss of PTRF results in a reduced number of caveolae and a dystrophic phenotype (17). Experiments using fluorescence lifetime imaging showed that cholesterol is required for the interaction between PTRF and caveolin. Since cholesterol depletion decreases the PTRF-caveolin interaction, this suggests that PTRF may be involved in stabilizing the membrane curvature of caveolae (62). Additionally, it was shown by immunoprecipitation that PTRF binds dysferlin and, in fact, may be required for the correct localization of dysferlin, since PTRF mutation results in decreased dysferlin at the cell membrane (17). Knockdown of PTRF by shRNA results in decreased membrane repair capacity, whereas overexpression of PTRF can rescue dystrophic muscle membrane repair (143). During the membrane repair process, PTRF binds to dysferlin and may anchor MG53/TRIM72 to cholesterol, since MG53/TRIM72 cannot bind cholesterol unless PTRF is present (92, 143). In this model, PTRF anchors MG53/TRIM72 by binding to exposed cholesterol at the membrane injury site (FIGURE 3).

Motor Proteins (Nonmuscle Myosin IIA and IIB/Kinesin)

Since vesicle trafficking is an important aspect of membrane repair, motor proteins must be involved in this process to allow trafficking to occur. Membrane repair is sensitive to inhibitors of both myosin and kinesin motor proteins (126, 131). Studies have specifically shown that non-muscle myosin IIA and B are important motor proteins that mediate vesicle trafficking during membrane repair (131). Antisense knockdown of myosin IIB suppressed exocytosis and membrane resealing, and knockdown of myosin IIA inhibited the rate of resealing at repeated wound sites. These studies are supported by the observation that non-muscle myosin IIA facilitates the transport of vesicles containing MG53/TRIM72 to the site of membrane injury (86) (FIGURE 3).

Affixin [β-Parvin/Integrin-Linked Kinase (ILK)-Binding Protein]

Affixin was first discovered to be an integrin-linked kinase binding protein that localizes to focal adhesions and likely contributes to their maturation. In muscle cells, affixin and ILK colocalize at sites where the Z band attaches to the sarcolemma due to an interaction with dysferlin (94). Affixin's interaction with dysferlin and altered immunoreactivity in MM, LGMD2B, and LGMD1C suggest a role for this protein in muscle membrane repair, possibly through organization of F-actin (FIGURE 2A) (94). For example, affixin has been shown by immunoprecipitation and pull-down assay to interact with guanine nucleotide exchange factor αPIX (ARHGEF6 or Cool-2), which functions to regulate actin skeleton adhesion (93, 94, 104) as well as α-actinin, which plays a role in organization of the cytoskeleton (142). Although it appears that affixin may participate in cytoskeletal remodeling during membrane repair, the precise role of affixin in this process remains speculative.

Acid Sphingomyelinase

Acid sphingomyelinase (ASM) is an enzyme that cleaves the phosphorylcholine head of sphingomyelin to generate ceramide, a molecule that leads to membrane invagination, and that contributes to the process of endocytosis during membrane repair (64, 124). In response to injury, lysosomes fuse with the injured cell membrane and release ASM from the cell. The action of ASM causes invagination of the membrane and endocytosis of the injury site (FIGURE 2B) (29, 35, 42, 129). Additional evidence of ASM contribution to membrane repair is provided by the finding that dysferlin-deficient C2C12 cells showed less secretion of ASM than control cells (35). When ASM was inhibited and cells were permeabilized using a pore-forming toxin, the cells could not sufficiently repair (35). Treatment with exogenous ASM was sufficient to restore the membrane integrity of ASM-depleted cells (35) and of a dystrophic patient-derived myoblast cell line (35).

ESCRT

Endosomal sorting complex required for transport (ESCRT) is involved in viral budding, cytokinesis, and spontaneous budding of the plasma membrane. ESCRT subunits are classified into five complexes (108). The ESCRT III complex recently has been shown to be involved in endocytosis and budding in response to membrane damage (30, 74, 122). After injury, apoptosis-linked gene (ALG)-2 binds Ca²⁺ and leads to the accumulation of ESCRT III and accessory proteins ALG-2-interacting protein X (ALIX), and vacuolar protein sorting-associated protein 4 (Vps4) (122). Assembly of this complex results in cleavage of the wounded membrane and shedding of the wound site into extracellular vesicles. In response to UV laser injury, ESCRT III is recruited to injury sites exercising calcium-dependent wound repair. The observed recruitment of ESCRT is followed by blebbing of the membrane and shedding of the wound (FIGURE 2B). ESCRT III is involved in endocytosis of the membrane after the insertion of the bacterial pore-forming toxin SLO (74). The complex appears to be critical for repair of injuries of <100 nm (74).

Annexin

Annexins are a protein family consisting of a COOH terminus containing phospholipid and Ca²⁺ binding sites, and a variable NH₂ terminus (111). The annexins are unique in that they can bind lipids while in their Ca²⁺-bound conformation, and the concentration of the Ca²⁺ signal determines which particular annexin family member will bind to the phospholipid (53). Although the exact mechanism remains unclear, numerous studies suggest roles for annexins in vesicle movement, fusion, and patch formation during membrane repair (54, 57, 95). Annexins A1 and A2 bind dysferlin in a Ca²⁺-dependent manner and may contribute to membrane repair by their ability to assist in the aggregation and fusion of intracellular vesicles by their association with lipid rafts of the plasma membrane (3, 85). Density gradient centrifugation experiments confirmed the Ca²⁺- and annexin-dependent association of these rafts. Electron microscopic evidence shows that annexin may be involved in membrane fusion during exocytosis as well as serving as a scaffold for endosomes (FIGURE 2C) (11, 53). Annexins also have been shown to mediate microvesicle release and blebbing, an indirect repair mechanism that involves sealing off a damaged membrane segment by accumulation of annexin at the neck of the membrane bleb (43). Other studies indicate that annexin A5 (AnxA5) can bind to injured membranes and form a two-dimensional array that is thought to contribute to membrane repair, since AnxA5-null cells show defective repair capacity (15). Finally, annexin A6 has been shown by live imaging to locate to membrane disruptions and to assemble into a “cap” on the membrane repair patch to assist with membrane resealing (128).

AHNAK (Desmoyokin)

AHNAK, or desmoyokin, is a 629.1-kDa tripartite nucleoprotein with potential functions as diverse as fat metabolism, DNA repair, autoantigenicity, cell signaling, calcium channel regulation, and tumor metastasis (33). It has been proposed that interaction of AHNAK with annexin 2 and S100A10 regulates organization of the actin cytoskeleton and architecture of the cell membrane, since AHNAK-specific siRNA prevents actin cytoskeleton reorganization (10). In membrane repair, AHNAK has been associated with enlargeosomes, vesicles that rapidly exocytose in response to calcium influx; however, its exact role within these vesicles is unknown (14). AHNAK's ability to bind actin may signify a role for the protein in membrane resealing, although its presence within enlargeosomes during recruitment to the membrane injury suggests earlier involvement in the repair process (33). The co-localization of AHNAK with annexin 2 within vesicles is controversial, as studies also have reported that annexin is on the cytosolic face of the vesicle (89). In fact, calcium-sensitive annexin 2 may be involved in recruitment of AHNAK and S100A10 to the plasma membrane in response to calcium-annexin binding (33). Furthermore, co-immunoprecipitation and mass spectrometry studies confirm that AHNAK interacts with dysferlin, which may localize and stabilize AHNAK at the sarcolemma through its transmembrane domain, an interaction possibly regulated by calpain 3 (67, 68) (FIGURE 2).

S100A10

S100A10 is a small, ∼10-kDa EF-hand Ca²⁺ binding protein also known as annexin 2 light chain or p11. S100A10 forms a heterotetrameric complex with annexin A2 by forming an S100A10 dimer in the middle of two annexin A2 chains. This complex targets to the plasma membrane in a calcium-regulated manner that is dependent on its interaction with annexin 2 (FIGURE 2C). At the plasma membrane, S100A10 interacts with a number of proteins, where it may be essential for surface presentation of proteins such as ion channels (117). Furthermore, proteomic and structural analyses have identified that the heterotetrameric complex binds cytosolic proteins AHNAK and dysferlin, and that it is responsible for the recruitment of AHNAK to the cell membrane, where the entire complex acts as a scaffold for membrane repair (38, 81, 111, 118). Although S100A10 is known to be a central player in the membrane repair complex, its exact function in membrane repair has not been identified. One model suggests that membrane fusion may occur due to the ability of S100A10-annexin A2 to bridge adjacent phospholipid membranes (54).

Synaptotagmin/SNAREs

Synaptotagmins (Syt) are a family of proteins that contain two C2 domains (C2A and C2B), which some members use to bind Ca²⁺ or phospholipids (16, 65, 102, 123, 127). Much of the focus on synaptotagmins has been in synaptic neurotransmitter release in neurons through their interactions with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) (13, 32, 46, 49, 51, 52, 103). The general mechanism of this interaction begins with vesicular SNAREs interacting with membrane SNAREs to dock the vesicle to the membrane. Synaptotagmins bound to the vesicle bind Ca²⁺ and are then able to interact with the SNAREs to fuse the vesicle to the membrane and expel the contents of the vesicle (FIGURE 2D). Membrane repair is not solely dependent on synaptotagmins because studies using botulinum toxin A, which cleaves SNARE proteins, showed inhibition of resealing of sea urchin eggs (12). Disruption of the formation of the SNARE complex using the cytoplasmic domain of synaptobrevin 2, a SNARE protein, was also able to block membrane resealing (125).

The relationship between synaptotagmins and SNAREs is important because of their roles in Ca²⁺ sensing and vesicle membrane fusion. Syt I is found exclusively in the nervous system (48, 138), and antibodies against the C2A domain of Syt I caused inhibition of membrane repair in severed axons of squid and crayfish giant axons (40). This same study used antibodies against a domain of a SNARE protein, syntaxin, to inhibit interaction with synaptotagmin's C2A domain (40). Similar to inhibition of Syt I, blocking syntaxin inhibited resealing of the severed axon (40). Syt VII is ubiquitously expressed and found proximal to the membrane on lysosomal-associated membrane protein 1 (LAMP-1)-positive lysosomes. Cells injured in the presence of Syt VII blocking antibodies showed a decreased capacity to reseal membrane disruptions (115), and fibroblasts taken from Syt VII-deficient mice showed defective lysosomal exocytosis and decreased capacity to reseal their plasma membrane (22).

Conclusions

We have attempted to summarize the available literature concerning the roles of many of the proteins known to contribute to the membrane repair process. While clear evidence for the involvement of the discussed proteins in membrane repair exists, it should be noted that other molecules, such as Amphiphysin 2 (BIN1) (113), Lamp-1 (115), and others, have been speculated to play a role in membrane resealing due to the nature of their actions in the cell or due to their interactions with known members of the membrane repair machinery. For example, a recent study identified ATPase EH-domain containing 2 as a novel membrane repair protein that co-localizes at the site of membrane injury with F-actin and annexin A1 (91). Moesin (membrane-organizing extension spike protein) has been shown to interact with dysferlin and appears to cross-link plasma membranes and actin cytoskeletons (17). As more information about specific proteins and membrane repair in general is obtained, we expect that additional candidates will be discovered.

In considering the molecules known to be important in the membrane repair process, it becomes clear that many of the steps in membrane repair are processes necessary for normal cellular functions. For example, myosin, kinesin, annexin, and SNAREs are involved in vesicle trafficking and membrane fusion, integral events for numerous cell activities (39). Calpain and affixin are important for cytoskeletal remodeling during cell motility, responses to the cell environment and mitosis (66, 120). PTRF, ESCRT, ASM, and S100A10 are active at the plasma membrane to form caveolae, membrane buds, and other membrane invaginations as well as to mediate membrane fusion (17, 54, 74, 129). Dysferlin is integral to multiple activities, such as endocytosis, vesicle trafficking, membrane turnover, and others (31, 47, 58). Membrane repair can be considered an emergency response in which these cellular processes are used to reseal the membrane and allow cell survival (57, 99, 105). However, it is necessary to move beyond dissection of this supportive cellular machinery to gain a full understanding of membrane repair. Given the current understanding in the field, it is difficult to define the prevailing membrane repair hypothesis, and it is likely that there is no single mechanism that is at work under all injury scenarios. Our current understanding of membrane repair is limited to a subset of cellular functions and protein interactions, leaving compelling questions unanswered. For example, how does the cell differentiate the membrane repair process from normal cell functions and what are the specific signaling pathways that allow this specific response to occur? Furthermore, what are the signals that direct the repair machinery to the site of injury? While calcium-dependent mechanisms are known signals for the assembly of membrane repair proteins at the damage site and for fusion of membrane surfaces, additional mediators may exist. Discovery and characterization of these mediators and pathways are important next steps in understanding the membrane repair process.