**4.1. Large wounds: exocytic repair, membrane patches and endocytic repair**

### *4.1.1. Oocytes*

**Cell type Repair**

Somatic cells Neuronal cells

**mechanism**

200 Wound Healing - New insights into Ancient Challenges

Actomyosin contractile ring

Synaptotagminmediated exocytosis

Calpainmediated vesicle fusion

mediated exocytosis

Facilitated resealing

Potentiated resealing

patch" formation; Dysferlinmediated exocytosis

Caveolaemediated endocytic repair

Caveolaemediated endocytosis

ESCRTmediated shedding

PFTs: Pore-forming toxins; ASM: Acid Sphingomyelinase

**Table 1.** Wound healing mechanisms according to cell-type.

Fibroblasts Synaptotagmin-

Muscle cells "Membrane

Epithelial cells

Synaptotagminmediated exocytosis and "vertex fusion"

**Experimental wound type(s)**

Mechanical wounding; laser wounding

Mechanical wounding; laser wounding

Axon transection

Mechanical wounding

Mechanical wounding; laser wounding

Mechanical wounding

**Major molecular players**

Yolk granules; synaptobrevin; synaptotagmin VII; SNAP-25

RhoA; Cdc42; F-actin; myosin II; microtubules

Lysosomes; synaptotagmin I; syntaxin I;

Lysosomes; synaptotagmin I; Synaptotagmin VII; VAMP-7; Syntaxin-4;

SNAP-23

Laser wounding PKG; CREB [99]

Lysosomes; calpains; mini-dysferlinC72; MG53; KIF5B; AHNAK; S100A10; Annexin II

ASM2

PFTs1 ASM; Caveolin-3;

PFTs ALG-2; ESCRT-III;

annexin I

annexin I

ALIX; Vps4

; Caveolin-3;

vesicles; myosin IIA; PKC

Laser wounding Predocked TGN-

Axon transection Calpains [52, 92–94]

**Reference(s)**

[76–82]

[83–89]

[90, 91]

[76, 77, 95]

[51, 96–98]

[50, 100–107]

[108–113]

[45, 108, 110]

[74, 114]

Oocytes of *Xenopus laevis* and sea urchins are large, easily accessible and manipulable cells. While their size and lack of adhesive and cell-cell contact-derived tension distinguish them from mammalian somatic cells, these characteristics also provide a simpler platform which helped to elaborate the first models of single-cell wound healing. Oocytes have been observed to recover form very large mechanical disruptions of both the plasma membrane and cytoskeleton (>1000 µm2 ) [75].

### *4.1.1.1. "Membrane patch"-mediated resealing*

In addition to providing essential amino acids and other nutrients for oocyte development, yolk platelets also act as vesicle reservoirs upon plasma membrane injury of oocytes in a variety of species [22, 75]. Upon wounding, there is a rapid influx of Ca2+ from the extracellular milieu to the intracellular space, which favors rapid homotypic fusions of yolk granules [75]. These homotypic vesicular fusogenic events lead to the formation of a large "membrane patch" that eventually covers the gap present at the wound site [75–77]. It is perhaps best to think of the "membrane patch" model of wound healing as a somewhat oocyte-specific process. Indeed, this model relies almost entirely on homotypic fusions [75], and yolk granules offer a pool of readily available vesicle reserves that is incomparable with those available to somatic cells [78]. Also, while the "membrane patch" model of single-cell repair has also initially been proposed for the repair of large wounds in somatic cells [79, 80], it now appears that large somatic cell membrane disruptions are directly removed by endocytosis, which heavily relies on exocytosis and heterotypic fusion events (reviewed in [35, 81]; see Section 4.1.3.2). Whether these apparent differences are an intrinsic property of oocytes, a direct consequence of the wound size involved, larger wounds exposing larger areas of the intracellular space to Ca2+, or of the higher density of available vesicles in oocytes, remains open for interpretation.

While the mechanism behind membrane patch formation is sufficient to block unregulated exchanges between extra- and intracellular spaces, it does not technically reseal the membrane, or restore membrane continuity or normal plasma membrane composition and shape. The way this resealing is achieved is still somewhat unclear and is the subject of two alternate but compatible models. The first states that heterotypic fusion events between intracellular vesicles, the membrane patch and the borders of the wounded plasma membrane first restore membrane continuity, after which contraction of an actomyosin ring restores normal plasma membrane composition and shape ([82]; see Section 4.1.1.2). The so-called "vertex fusion" model relies on the same heterotypic fusion events but states that multiple fusion pores would form around the periphery of the wounded region. Expansion of these fusion pores may cause shedding of a membrane fragment containing both wound residual portions of the patch vesicle, in a mechanism reminiscent of the one observed for yeast vacuoles ([83, 84]; reviewed in [81]).

### *4.1.1.2. Actomyosin contractile ring*

As previously discussed, disruptions of the plasma membrane may be accompanied by, as well as induce direct and indirect cytoskeletal disruptions. Restoration of the local cytoskeleton is primarily driven by the contraction of a purse-string structure primarily assembled from Factin and myosin II [82]. This actomyosin ring is anchored to the plasma membrane at frequent points along its border [82], and its closure has been shown to restore normal membrane composition and shape [82].

Formation of the actomyosin array is controlled by the Ca2+-dependent recruitment and activation of Rho family GTPase proteins [85]. In *Xenopus* oocytes, activated Rho GTPases Ras homolog family member A (RhoA) and cell division control protein 42 homolog (Cdc42) localize to exclusive, concentric zones around the wound [86, 87]. These GTPases influence the activities of, among many other downstream targets, myosin light-chain kinase (MLCK) and myosin phosphatase [88, 89]. Through the above effectors, RhoA indirectly regulate the phosphorylation levels of myosin II light chains, mediating the assembly and contraction of the actomyosin ring (reviewed in [85]). As for Cdc42, its interactions with neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein 2/3 (Arp2/3) [90–92] induce construction of highly dynamic, branched F-actin networks [86]. Binding of Arp2/3 with the C terminus of N-WASP, which is activated by Cdc42, stimulates Arp2/3's actin nucleation activity [91], accelerating production of actin networks critically involved in actomyosin ring assembly. The formation of contractile arrays has been demonstrated to also be regulated by an underlying "signaling treadmill" [93] in which gradients of Rho GTPase activities influence F-actin turnover. RhoA is preferentially activated and maintains its zone of high activity at the leading edge of the wound [93], while active Cdc42 encircles the inner RhoA zone [86]. The processes leading to the establishment of these concentric zones is still somewhat unclear, but a recent study by Vaughan et al. [94] has led to some interesting insights. They observed that wounding induced the formation of micrometer-scale PIP2−, phosphatidylinositol 3,4,5 trisphosphate (PIP3)- and phosphatidylserine (PS)-, phosphatidic acid (PA)- and diacylglycerol (DAG)-enriched domains. This is of particular interest as PS moved to a zone closest to the wound edge, near to an area of high RhoA activity, whereas PIP2 and PIP3 were observed to be associated with the so-called Cdc42 zone. As for DAG and PA, both of them were shown to immediately segregate in a zone overlapping that of which of RhoA and Cdc42 activity. Since DAG is known to be able to recruit PKCβ and PKCη [95], the authors suggested that generation of DAG at the wound site could therefore act as an upstream signal for the regulation of RhoA and Cdc42. Whether a similar signal cascade exist for somatic cells is still unclear, but celllifting experiments done of primary epithelial cells induced phospholipase D (PLD) activation was transient, consistent with a possible role in membrane repair and PLD inhibitors inhibited membrane resealing upon laser injury [96].

Whether such a contractile ring can form in the smaller wounds associated with somatic cells is unclear, but the formation of strikingly similar concentric zones of Rho1, Cdc42 and Rac have been shown to form in *Drosophila* syncytial embryos following plasma membrane wounding (reviewed in [97]).

### *4.1.2. Neuronal cells and fibroblasts: insights into exocytic repair*

this model relies almost entirely on homotypic fusions [75], and yolk granules offer a pool of readily available vesicle reserves that is incomparable with those available to somatic cells [78]. Also, while the "membrane patch" model of single-cell repair has also initially been proposed for the repair of large wounds in somatic cells [79, 80], it now appears that large somatic cell membrane disruptions are directly removed by endocytosis, which heavily relies on exocytosis and heterotypic fusion events (reviewed in [35, 81]; see Section 4.1.3.2). Whether these apparent differences are an intrinsic property of oocytes, a direct consequence of the wound size involved, larger wounds exposing larger areas of the intracellular space to Ca2+, or of the higher

While the mechanism behind membrane patch formation is sufficient to block unregulated exchanges between extra- and intracellular spaces, it does not technically reseal the membrane, or restore membrane continuity or normal plasma membrane composition and shape. The way this resealing is achieved is still somewhat unclear and is the subject of two alternate but compatible models. The first states that heterotypic fusion events between intracellular vesicles, the membrane patch and the borders of the wounded plasma membrane first restore membrane continuity, after which contraction of an actomyosin ring restores normal plasma membrane composition and shape ([82]; see Section 4.1.1.2). The so-called "vertex fusion" model relies on the same heterotypic fusion events but states that multiple fusion pores would form around the periphery of the wounded region. Expansion of these fusion pores may cause shedding of a membrane fragment containing both wound residual portions of the patch vesicle, in a mechanism reminiscent of the one observed for yeast vacuoles ([83, 84]; reviewed

As previously discussed, disruptions of the plasma membrane may be accompanied by, as well as induce direct and indirect cytoskeletal disruptions. Restoration of the local cytoskeleton is primarily driven by the contraction of a purse-string structure primarily assembled from Factin and myosin II [82]. This actomyosin ring is anchored to the plasma membrane at frequent points along its border [82], and its closure has been shown to restore normal membrane

Formation of the actomyosin array is controlled by the Ca2+-dependent recruitment and activation of Rho family GTPase proteins [85]. In *Xenopus* oocytes, activated Rho GTPases Ras homolog family member A (RhoA) and cell division control protein 42 homolog (Cdc42) localize to exclusive, concentric zones around the wound [86, 87]. These GTPases influence the activities of, among many other downstream targets, myosin light-chain kinase (MLCK) and myosin phosphatase [88, 89]. Through the above effectors, RhoA indirectly regulate the phosphorylation levels of myosin II light chains, mediating the assembly and contraction of the actomyosin ring (reviewed in [85]). As for Cdc42, its interactions with neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein 2/3 (Arp2/3) [90–92] induce construction of highly dynamic, branched F-actin networks [86]. Binding of Arp2/3 with the C terminus of N-WASP, which is activated by Cdc42, stimulates Arp2/3's actin nucleation activity [91], accelerating production of actin networks critically involved in actomyosin ring

density of available vesicles in oocytes, remains open for interpretation.

in [81]).

*4.1.1.2. Actomyosin contractile ring*

202 Wound Healing - New insights into Ancient Challenges

composition and shape [82].

Neuronal cells have markedly polarized membranes, with extreme distances between axons and the cell soma. The elongated morphology of axons make them particularly susceptible to shear stress injury and offering a challenge to vesicle trafficking. Fibroblasts, on the other hand, offer a relatively simpler platform for the study of single-cell wound healing.

While the repair of oocytes relies on homotypic fusion of abundant yolk granules (see Section 4.1.1.1), repair of mammalian cells has long been observed to depend on the Ca2+-dependent exocytosis of intracellular vesicles [98]. Conventional lysosomes are not only the major vesicles responsible for Ca2+-dependent exocytosis in non-neuronal and non-secretory cells [99], but also occupy a central role in the exocytic [100] and endocytic models of single-cell repair (see Section 4.1.3.2). The lysosomes involved in plasmalemma repair can be defined as lysosomalassociated membrane protein 1 (LAMP-1)-positive [100], acid sphingomyelinase (ASM) containing intracellular vesicles [101].

Exocytosis-mediated repair attracted considerable interest when Steinhardt et al. [98] specified the mechanistic similarities with Ca2+-triggered synaptic exocytosis, both of which are dependent on Ca2+ [102, 103] and actin cytoskeleton dynamics [104–106]. Ca2+-triggered synaptic exocytosis involves the interaction of synaptotagmin I, a C2 domains-containing protein present in exocytic vesicles and the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes of the synaptic membrane (reviewed in [107]). Interestingly, neurotransmission inhibitors botulinum neurotoxin A and B also negatively affected or completely blocked membrane healing in sea urchin embryos and Swiss 3T3 fibroblasts [98]. Similarly, treatment with an antibody targeting the active synaptotagmin I C2A domain was observed to prevent membrane resealing of squid and crayfish giant axons [108], 3T3 fibroblasts [100] and rat PC12 cells [109]. In contrast to the neuron-specific synaptotagmin I [110], synaptotagmin VII is ubiquitously expressed and has been found to also influence exocytic membrane repair of other cell types such as sea urchin embryos [98, 103], Chinese hamster ovary cells [100], Swiss 3T3 fibroblasts [98, 100, 111], mouse embryonic fibroblasts [112] and epithelial cells [113–115]. Indeed, embryonic fibroblasts of synaptotagmin VIIdeficient mice were observed to have defects in lysosome exocytosis and wound resealing [112]. The mechanism involves the Ca2+-dependent activation and recruitment of synaptotagmin VIII-positive vesicles [111], which are then transported to the wound site via microtubuledependent trafficking [116]. Once at the plasma membrane, lysosome-bound synaptotagmin VII interacts with the SNARE formed by vesicle-associated membrane protein 7 (VAMP-7), syntaxin-4 and synaptosomal-associated proteins (SNAPs), such as SNAP-23, which leads to heterologous fusion of the lysosome with the plasma membrane [115].

As opposed to the "membrane-patch" model that relies on predominantly homotypic fusions, the exocytic model of single-cell repair assumes the predominance of heterotypic fusion events with the plasma membrane. Indeed, early microneedle-wounding experiments clearly showed a punctate distribution of lysosomal marker LAMP-1 around the wound site [100]. These heterotypic fusion events were initially thought to promote resealing by increasing plasma membrane surface area [100, 117], thereby lowering in-plane membrane tension (see Section 3.1), which could theoretically favor spontaneous resealing events between the two wound edges of with nearby vesicles [118]. Indeed, inhibiting the lowering of in-plane membrane tension via actin stabilization, or by inhibiting exocytosis via neurotoxins A and B ([51, 118] inhibits successful cell repair but is rescued by artificially reducing in-plane tension via the addition of surface active Pluronic F68 NF [51]).

As previously stated, the in-plane tension lowering effect of exocytosis indubitably has been shown to be crucial for plasma membrane repair of a variety of wounds (reviewed in [35, 119]), several lines of evidence have shown that they are not the last process in the resealing of plasma membranes. Indeed, aside from the notable exceptions of facilitated membrane repair that involves recruitment of additional vesicles originating from the transgolgi network (TGN) [120], there is little to no evidence that large micrometer size wounds are repaired via purely exocytic means. Rather, it seems that exocytosis acts as a preliminary step of other wound-healing process (see Section 4.1.3.2). In fact, there is debate as to whether conclusions made in earlier studies of exocytosis were misinterpretations of endocytic vesicles as an exocytic patch or vesicles, due to the studies being performed in the absence of extracellular endocytic tracers [121]. Similarly, there is also considerable evidence that the synaptotagmin VII/SNARE system may not be the only, or even the main, fusogenic system that mediates Ca2+-dependent exocytosis following injury, at least in cells that are under constant mechanical assault such as muscle fibers and muscle cells (see Section 4.1.3).

### *4.1.3. Muscle cells*

attachment protein receptor (SNARE) complexes of the synaptic membrane (reviewed in [107]). Interestingly, neurotransmission inhibitors botulinum neurotoxin A and B also negatively affected or completely blocked membrane healing in sea urchin embryos and Swiss 3T3 fibroblasts [98]. Similarly, treatment with an antibody targeting the active synaptotagmin I C2A domain was observed to prevent membrane resealing of squid and crayfish giant axons [108], 3T3 fibroblasts [100] and rat PC12 cells [109]. In contrast to the neuron-specific synaptotagmin I [110], synaptotagmin VII is ubiquitously expressed and has been found to also influence exocytic membrane repair of other cell types such as sea urchin embryos [98, 103], Chinese hamster ovary cells [100], Swiss 3T3 fibroblasts [98, 100, 111], mouse embryonic fibroblasts [112] and epithelial cells [113–115]. Indeed, embryonic fibroblasts of synaptotagmin VIIdeficient mice were observed to have defects in lysosome exocytosis and wound resealing [112]. The mechanism involves the Ca2+-dependent activation and recruitment of synaptotagmin VIII-positive vesicles [111], which are then transported to the wound site via microtubuledependent trafficking [116]. Once at the plasma membrane, lysosome-bound synaptotagmin VII interacts with the SNARE formed by vesicle-associated membrane protein 7 (VAMP-7), syntaxin-4 and synaptosomal-associated proteins (SNAPs), such as SNAP-23, which leads to

As opposed to the "membrane-patch" model that relies on predominantly homotypic fusions, the exocytic model of single-cell repair assumes the predominance of heterotypic fusion events with the plasma membrane. Indeed, early microneedle-wounding experiments clearly showed a punctate distribution of lysosomal marker LAMP-1 around the wound site [100]. These heterotypic fusion events were initially thought to promote resealing by increasing plasma membrane surface area [100, 117], thereby lowering in-plane membrane tension (see Section 3.1), which could theoretically favor spontaneous resealing events between the two wound edges of with nearby vesicles [118]. Indeed, inhibiting the lowering of in-plane membrane tension via actin stabilization, or by inhibiting exocytosis via neurotoxins A and B ([51, 118] inhibits successful cell repair but is rescued by artificially reducing in-plane tension via the

As previously stated, the in-plane tension lowering effect of exocytosis indubitably has been shown to be crucial for plasma membrane repair of a variety of wounds (reviewed in [35, 119]), several lines of evidence have shown that they are not the last process in the resealing of plasma membranes. Indeed, aside from the notable exceptions of facilitated membrane repair that involves recruitment of additional vesicles originating from the transgolgi network (TGN) [120], there is little to no evidence that large micrometer size wounds are repaired via purely exocytic means. Rather, it seems that exocytosis acts as a preliminary step of other wound-healing process (see Section 4.1.3.2). In fact, there is debate as to whether conclusions made in earlier studies of exocytosis were misinterpretations of endocytic vesicles as an exocytic patch or vesicles, due to the studies being performed in the absence of extracellular endocytic tracers [121]. Similarly, there is also considerable evidence that the synaptotagmin VII/SNARE system may not be the only, or even the main, fusogenic system that mediates Ca2+-dependent exocytosis following injury, at least in cells that are under constant mechanical

heterologous fusion of the lysosome with the plasma membrane [115].

addition of surface active Pluronic F68 NF [51]).

204 Wound Healing - New insights into Ancient Challenges

assault such as muscle fibers and muscle cells (see Section 4.1.3).
