**4.2. Small wounds: ectocytic repair, blebbing and membrane shedding**

### *4.2.1. Ectocytic repair of pore-forming toxins and blebbing of small mechanical wounds*

As previously stated, caveolae-mediated endocytosis was shown to mediate the repair of membrane disruptions created by pore-forming toxins [45]. Indeed, SLO was directly visualized entering cells within caveolar vesicles [153]. Post-internalization, the pore has been shown to be ubiquitinated and eventually degraded by lysosomal hydrolysis [45].

While endocytic repair of SLO pores is now a widely accepted mechanism, it still raises some questions as SLO pores were shown to be successfully removed from the neurites of SH-SY5Y neuroblastoma cells [162]. SH-SY5Y cells are devoid of lysosomes and hence cannot undergo caveolae-mediated endocytosis.

As discussed in Section 3.3.1, Ca2+ entry can lead to local actin depolymerization, which in turn leads to a diminution of apparent membrane tension, and the formation of membrane blebs [163]. Indeed, bleb formation seems to principally depend on osmotic pressure and MCAs, the contribution of in-plane tension being minimal [164, 165]. Formation of membrane blebs can be initiated by laser ablation of the cortex cytoskeletal structures [163]. This also explains why formation of membrane blebs is inhibited by drugs that leads to depolymerization [166] or stabilization [167] of the actin cytoskeleton.

SLO pores cause localized Ca2+ entry and actin depolymerization without creating large plasma membrane tears-related increases in membrane tension (see Section 3.3.1). As such, it is not surprising that small blebs may be involved in the removal of SLO pores [168]. It should be noted that the same observations strongly suggest that SLO pore insertion, pore assembly, pore clustering and even bleb formation may be Ca2+-independent [168]. This is controversial, however, as it would imply that SLO pore insertion could possibly displace proteins responsible of the interaction of the plasmalemma with the cytoskeleton. This also poses a problem, as other teams have shown a Ca2+ [169] and actin disruption [162] dependence for the survival of SLO-treated cells [169], and for the shedding of SLO-laden microvesicles [162]. In this alternate model [162], pore disruption of the membrane elevates local Ca2+ concentration which in turn activates annexins and calpains. Calpains then disrupt the underlying actin cytoskeleton, thereby facilitating bleb formation [170] and shedding of SLO vesicles.

### *4.2.2. ESCRT-mediated shedding of small disruptions*

The endosomal sorting complex required for transport (ESCRT) complexes are factors of the lysosomal pathway during protein processing and are involved in various membrane remodeling events such as lysosomal targeting of ubiquitinated proteins and multivesicular body biogenesis, as well as cytokinetic abscission ([171]; reviewed by Olmos and Carlton [172]). There are five currently known ESCRT complexes: ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and ESCRT-IV. Of these, ESCRT-III has since been found to modulate much of the membrane remodeling processes, while ESCRT-0, ESCRT-I and ESCRT-II facilitate its targeting to specific cellular compartments, ESCRT-IV orchestrating the disassembly of the ESCRT-III complex for subunit recycling (reviewed in [173]). An additional function of ESCRT-III in plasma membrane repair was proposed in a recent study, which suggested that ESCRT-III is involved in the pinching out or shedding of wounded membranes in HeLa cells [74]. Indeed, injuryinduced Ca2+ increase results in Ca2+ binding of apoptosis-linked gene-2 (ALG-2) around the site of disruption. Active ALG-2 initiates ESCRT machinery assembly by facilitating the accumulation of ALG-2-interacting protein X (ALIX) near the wound site, after which ALG-2 and ALIX recruit ESCRT-III and vacuolar protein sorting-associated protein 4 (Vps4) to the injured plasma membrane [74, 174]. These subunits form a complex, which cleave and shed the wound from the plasma membrane to extracellular space [174]. ESCRT-mediated shedding leads to a decrease in total plasma membrane surface area, increasing in-plane membrane tension.

### **5. Conclusion**

Injury-induced disruptions to the plasma membrane's shape and composition directly affect the cell's tensegrity. The different active membrane repair mechanisms that have been discussed in this chapter are perhaps best seen as a single interconnected pathway, in which the type and size of the wound determine the extent and severity of factors such as tension change and Ca2+ entry. These factors in turn dictate the healing mechanisms being used (**Table 1** and **Figure 2**). Mechanical lesions lead to high, localized levels of membrane integrity loss, tension change and Ca2+ influx. These physical tears of the plasma membrane are often repaired by targeted exocytosis and endocytosis. Contrastingly, smaller injuries such as those generated by electroporation and osmotic shock induce low levels of membrane disruption, tension change and Ca2+ influx across large membrane areas. These in turn facilitate processes such as cytoskeletal remodeling or caveolae flattening. Conversely, membranes disrupted by toxic pores do not lead to substantial increase in plane tension. As such, they can either be rapidly shed or degraded following caveolae-mediated endocytosis. Furthermore, it appears that the wound-healing mechanisms prevalent in a given cell-type fall not only in accordance with the prevalence of specific injury types (i.e., PFTs vs. tears vs. ablations), but also according to cell type-specific differences in cell tensegrity and polarity (e.g., muscle cells vs. epithelial cells).

SLO pores cause localized Ca2+ entry and actin depolymerization without creating large plasma membrane tears-related increases in membrane tension (see Section 3.3.1). As such, it is not surprising that small blebs may be involved in the removal of SLO pores [168]. It should be noted that the same observations strongly suggest that SLO pore insertion, pore assembly, pore clustering and even bleb formation may be Ca2+-independent [168]. This is controversial, however, as it would imply that SLO pore insertion could possibly displace proteins responsible of the interaction of the plasmalemma with the cytoskeleton. This also poses a problem, as other teams have shown a Ca2+ [169] and actin disruption [162] dependence for the survival of SLO-treated cells [169], and for the shedding of SLO-laden microvesicles [162]. In this alternate model [162], pore disruption of the membrane elevates local Ca2+ concentration which in turn activates annexins and calpains. Calpains then disrupt the underlying actin cytoske-

The endosomal sorting complex required for transport (ESCRT) complexes are factors of the lysosomal pathway during protein processing and are involved in various membrane remodeling events such as lysosomal targeting of ubiquitinated proteins and multivesicular body biogenesis, as well as cytokinetic abscission ([171]; reviewed by Olmos and Carlton [172]). There are five currently known ESCRT complexes: ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and ESCRT-IV. Of these, ESCRT-III has since been found to modulate much of the membrane remodeling processes, while ESCRT-0, ESCRT-I and ESCRT-II facilitate its targeting to specific cellular compartments, ESCRT-IV orchestrating the disassembly of the ESCRT-III complex for subunit recycling (reviewed in [173]). An additional function of ESCRT-III in plasma membrane repair was proposed in a recent study, which suggested that ESCRT-III is involved in the pinching out or shedding of wounded membranes in HeLa cells [74]. Indeed, injuryinduced Ca2+ increase results in Ca2+ binding of apoptosis-linked gene-2 (ALG-2) around the site of disruption. Active ALG-2 initiates ESCRT machinery assembly by facilitating the accumulation of ALG-2-interacting protein X (ALIX) near the wound site, after which ALG-2 and ALIX recruit ESCRT-III and vacuolar protein sorting-associated protein 4 (Vps4) to the injured plasma membrane [74, 174]. These subunits form a complex, which cleave and shed the wound from the plasma membrane to extracellular space [174]. ESCRT-mediated shedding leads to a decrease in total plasma membrane surface area, increasing in-plane membrane

Injury-induced disruptions to the plasma membrane's shape and composition directly affect the cell's tensegrity. The different active membrane repair mechanisms that have been discussed in this chapter are perhaps best seen as a single interconnected pathway, in which the type and size of the wound determine the extent and severity of factors such as tension change and Ca2+ entry. These factors in turn dictate the healing mechanisms being used (**Table 1** and

leton, thereby facilitating bleb formation [170] and shedding of SLO vesicles.

*4.2.2. ESCRT-mediated shedding of small disruptions*

208 Wound Healing - New insights into Ancient Challenges

tension.

**5. Conclusion**

Similar to the plasma membrane and cytoskeletal elements interact to create tensegrity in the single-cell scale, adhesive forces of single cells and the extracellular matrix (ECM) provide structural stiffness to tissues [1]. Considering the above, it should be no surprise that successful single-cell repair influences the success of tissue repair. Indeed, contrary to tissue repair, singlecell repair is largely a binary event: it either takes place allowing the cell's survival, or not, leading to lysis or apoptotic removal. While relevant to wound healing at the tissue-level, these events have little to no relevance for single-cell wound healing outside of the modification of the environment of other injured cells in the surrounding area (asymmetric binding, change in ROS, Ca2+ concentration, etc.). Conversely, it seems that successful repair in one cell may lead to an increased repair potential in surrounding cells [175, 176]. This "potentiated" repair has been shown to involve purinergic and nitric oxide (NO)/PKG-signaling pathways [175, 176]. Similarly, repeated insults to a cell's structural and membrane integrity presumably affect a cell's ability to undergo subsequent membrane resealing and cytoskeletal repair, which would be reflected in its long-term viability in a given tissue. Indeed, the prominent view of the origin of the phenotypes associated with muscular dystrophies point toward a heighted susceptibly to repeated mechanical wounding, leading in turn in a higher rate of single-cell repair failure (reviewed in [177, 178]).

Another parallel between single-cell and tissue wound-healing mechanisms is their reliance on contractile arrays. This similarity has been confirmed in multicellular models such as *Xenopus* embryos [179], Caco-2 intestinal epithelial monolayers [180] and Madin-Darby canine kidney (MDCK) epithelial monolayers [181].

Wound closure in epithelial sheets has been demonstrated to be driven by the coupling of actomyosin contraction and collective cell migration [182–185]. The relative contribution of each mechanism in overall re-epithelialization depends on numerous biomechanical factors, including wound geometry [182–184], wound size [182, 186], tissue stiffness [186] and ECM composition [182]. In particular, wounds of cultured bovine corneal endothelial cell monolayers in ECM-deprived conditions were observed to reseal predominantly through actomyosin activity [182]. This is intriguing since cytoskeletal dynamics greatly influence single-cell wound-healing processes (see Section 3.3) and exhibits the ECM and cytoskeleton's analogous relationship across biological scales in the context of wound healing. These observations suggest that the importance of tensegrity components in wound repair are conserved across single-cell and multicellular models.

Considering the single cell's tensegral context in future wound-healing study will help further characterize an increasingly complex unified pathway theory of plasma membrane repair.
