**3.3. Cytoskeletal and MCA dynamics and wound healing**

The cytoskeleton constitutes a substantial component of apparent membrane tension in eukaryotic cells through MCAs (**Figure 1**). Consequently, cortical cytoskeleton dynamics can also reduce apparent membrane tension and constitutes an important preliminary step of single-cell wound healing. Indeed, actin destabilization has been demonstrated to enhance active membrane resealing in a variety of cell types, including 3T3 fibroblasts [51], septal neurons [52] and RGM1 gastric epithelial cells [53].

### *3.3.1. Direct and indirect regulation of single-cell injury by cytoskeleton dynamics*

Considering actin's importance for wound healing, it is not surprising that cellular injury affects actin dynamics in several ways. Changes of tensegrity experienced by damaged cells may lead to cytoskeletal remodeling either directly or through mechanotransductive signals. Indeed, sonoporation experiments showed that disruptions of existent plasmalemmal and adjacent cytoskeletal structures were enough to elicit a sustained and broad secondary disruption of the actin cytoskeleton [54]. As previously stated, actin filament bundles are the main providers of tensile forces necessary for a cell's tensegrity ([1]; **Figure 1**). Cells usually respond to external changes in tensile forces by modulating the sizes, numbers and distributions of F-actin and stress fibers in order to preserve mechanical homeostasis (reviewed in [55]). This is exemplified by experiments performed on endothelial cells [56] and osteoblasts [57] in which compression-induced stress fiber collapse through buckling, followed by actin disassembly events [56, 58]. Computer-assisted modeling strongly suggests that the loss of tensile force within the actin fiber upon its buckling is sufficient to induce actin disassembly [59, 60]. Whether a similar phenomenon contributes to actin fiber disassembly following mechanical damage is intriguing, as it would mean that actin filaments are able to act as their own mechanosensor. Indeed, a series of experiments showed that the tension state of individual actin filaments were inversely proportional to the binding affinity and actin filament-severing activity of cofilin [61–63]. Cofilin is an actin-binding protein that is known to accelerate actin depolymerization at the pointed end, which is also able to sever F-actin [64, 65]. This type of mechanosensing is especially attractive in the context of single-cell wound healing, as it is more sensitive and could induce downstream signals much faster than other traditional mechanosensors such as mechanosensitive ion channels [66], integrins, talin, or other F-actin-localized mechanosensors (reviewed in [62]).

Aside from mechanically related disruptions, cortical and cytoskeletal actin filaments are also disrupted in a variety of Ca2+-dependent manners. Indeed, permeabilization of cells by bacterial pores, such as streptolysin O (SLO), leads to an increase in intracellular Ca2+ without substantial direct damage to the plasmalemma or subjacent actin cytoskeleton and also incites actin depolymerization [67]. While Ca2+ is able to disrupt actin filaments on its own [68, 69], the effect of Ca2+ on the disruption of normal cytoskeletal architecture is probably best exemplified by its activation of calpains. Calpains are Ca2+-dependent, intracellular cysteine proteases that are known for their relative specificity [70]. Among others, calpains have been shown to cleave talin [71] into a large globular head domain that directly binds integrins, PIP2 and focal adhesion kinases, and a rod domain that binds vinculin and actin. Its degradation by calpains upon wounding would therefore be compatible with the cytoskeletal remodeling that follows membrane disruptions.

### *3.3.2. Cytoskeletal dynamics is at the center of single-cell wound healing processes*

active membrane resealing in a variety of cell types, including 3T3 fibroblasts [51], septal

Considering actin's importance for wound healing, it is not surprising that cellular injury affects actin dynamics in several ways. Changes of tensegrity experienced by damaged cells may lead to cytoskeletal remodeling either directly or through mechanotransductive signals. Indeed, sonoporation experiments showed that disruptions of existent plasmalemmal and adjacent cytoskeletal structures were enough to elicit a sustained and broad secondary disruption of the actin cytoskeleton [54]. As previously stated, actin filament bundles are the main providers of tensile forces necessary for a cell's tensegrity ([1]; **Figure 1**). Cells usually respond to external changes in tensile forces by modulating the sizes, numbers and distributions of F-actin and stress fibers in order to preserve mechanical homeostasis (reviewed in [55]). This is exemplified by experiments performed on endothelial cells [56] and osteoblasts [57] in which compression-induced stress fiber collapse through buckling, followed by actin disassembly events [56, 58]. Computer-assisted modeling strongly suggests that the loss of tensile force within the actin fiber upon its buckling is sufficient to induce actin disassembly [59, 60]. Whether a similar phenomenon contributes to actin fiber disassembly following mechanical damage is intriguing, as it would mean that actin filaments are able to act as their own mechanosensor. Indeed, a series of experiments showed that the tension state of individual actin filaments were inversely proportional to the binding affinity and actin filament-severing activity of cofilin [61–63]. Cofilin is an actin-binding protein that is known to accelerate actin depolymerization at the pointed end, which is also able to sever F-actin [64, 65]. This type of mechanosensing is especially attractive in the context of single-cell wound healing, as it is more sensitive and could induce downstream signals much faster than other traditional mechanosensors such as mechanosensitive ion channels [66], integrins, talin, or other F-actin-localized

Aside from mechanically related disruptions, cortical and cytoskeletal actin filaments are also disrupted in a variety of Ca2+-dependent manners. Indeed, permeabilization of cells by bacterial pores, such as streptolysin O (SLO), leads to an increase in intracellular Ca2+ without substantial direct damage to the plasmalemma or subjacent actin cytoskeleton and also incites actin depolymerization [67]. While Ca2+ is able to disrupt actin filaments on its own [68, 69], the effect of Ca2+ on the disruption of normal cytoskeletal architecture is probably best exemplified by its activation of calpains. Calpains are Ca2+-dependent, intracellular cysteine proteases that are known for their relative specificity [70]. Among others, calpains have been shown to cleave talin [71] into a large globular head domain that directly binds integrins, PIP2 and focal adhesion kinases, and a rod domain that binds vinculin and actin. Its degradation by calpains upon wounding would therefore be compatible with the cytoskeletal

*3.3.1. Direct and indirect regulation of single-cell injury by cytoskeleton dynamics*

neurons [52] and RGM1 gastric epithelial cells [53].

198 Wound Healing - New insights into Ancient Challenges

mechanosensors (reviewed in [62]).

remodeling that follows membrane disruptions.

Aside from its role in reducing apparent membrane tension, cytoskeleton remodeling is further required for single-cell wound healing as several plasma-resealing processes involve exocytosis of various vesicles such as lysosomes, MG53-positive vesicles and AHNAK-positive vesicles (reviewed in [35]; see Sections 4.1.2 and 4.1.3.1). As such, these intracellular vesicles must undergo actin- or microtubule-mediated transport to the wound site. An intact cortical cytoskeleton would therefore hinder not only transport and fusion of these vesicles, but also the subsequent removal of the damaged portions of the plasma membrane through endocytosis, blebbing or membrane-shedding processes. However, it should be noted that active repair mechanisms, such as exocytosis, cannot occur with just a minimal actin structure [67]. Reorganization of the cytoskeleton needs to be balanced in such a way that vesicles from intracellular pools are able to cross the actin barrier layer [53], then undergo docking to the wound site facilitated by remaining actin filaments [72] and the kinesin and myosin motor proteins [73].
