**3.2. Protein array-mediated wound site stabilization**

nor erythrocyte ghosts possess the machinery necessary to actively respond to the dramatic loss of tensegrity and changes in localized tensions that are created by large membrane disruption. Consequently, large erythrocyte ghosts' wounds do not spontaneously reseal under physiological conditions [14]. This has been attributed to a number of factors, including

Finally, plasma membrane disruption also exposes the cell to high levels of ROS and Ca2+ ions, either of which can be detrimental to normal cell function. Numerous pathways involving membrane dynamics such as the capacitation [23] and acrosomal reaction [24] steps of sperm maturation (reviewed in [25]) involve Ca2+-dependent signaling. Exocytosis events, such as surfactant secretion [26–28], as well as neuroendocrine [29], synapses [30–32] and auditory cells exocytosis [33, 34], are similarly Ca2+ dependent. These events are mediated by a variety of Ca2+-binding proteins such as calpains, annexins and synaptotagmins. Unsurprisingly, the uncontrolled Ca2+ entry that accompanies plasma membrane damage has been shown to activate the same families of Ca2+-binding proteins (reviewed in [35]). The downstream effects of Ca2+ entry will eventually lead to an overall diminution of apparent membrane tension.

the presence of strong MCAs [19] and the lack of endomembranes [22] (**Figure 1**).

196 Wound Healing - New insights into Ancient Challenges

**3. Early events of single-cell wound healing are mechanically driven**

tosis-, endocytosis- or membrane-shedding-mediated wound-healing processes.

**3.1. Caveolae-mediated decrease of in-plane membrane tension**

Single-cell repair proper is an active process, requiring dynamic and concerted manipulations of the cell's membrane and cytoskeletal compartments. Most of these processes, however, take place relatively late following injury or are dependent on preliminary disruption of cytoskeletal structures. In this subchapter, we present the principal wound mitigation events that are activated in the moments immediately following injury and facilitate the subsequent exocy-

Caveolae are plasma membrane invaginations with a diameter of 50–80 nm and specific flasklike morphology [36]. Caveolae have long been known to flatten in response to mechanically induced membrane deformation stretch [37]. Indeed, the preincubation of cells with methylβ-cyclodextrin diminishes the time to cell lysis upon hypotonic challenge [38]. Methyl-βcyclodextrin is a cholesterol-depleting compound that has also been shown to severely reduce the number of caveolae at the cell surface [39], probably by limiting the recruitment of caveolin oligomers to the plasma membrane. Caveolae can thus be viewed as a "membrane buffer" that limits injury-induced increases in apparent membrane tension by diminishing the in-plane tension without the need of additional membrane components from Ca2+-dependent exocytosis (**Figure 1**). Instead, additional membrane area is produced by the rapid flattening and disassembling of caveolae upon mechanical stress, which are rapidly reassembled upon

The exact molecular events leading to caveolae assembly and disassembly is still somewhat unclear, the specifics of which go beyond the scope of this chapter. Briefly, their assembly is

**processes**

mechanical stress release [40].

Most of the wound-healing mechanisms described to date (see Section 4) require a substantial lowering of apparent membrane tension. This is achieved in a number of ways, including the disruptions of MCAs through both Ca2+-dependent and Ca2+-independent membrane repair mechanisms (see Section 4). These Ca2+- and mechanosensor-mediated disruption of the MCAs and cytoskeletons have been shown to occur in large areas surrounding the wounds or throughout the cell and therefore only help to stabilize the wound indirectly.

The annexins form a large family of Ca2+-sensitive, negatively charged phospholipid-binding proteins (reviewed in [46]). Upon wounding, annexin V translocate to the internal leaflet of the damaged membrane where it binds to the newly exposed phosphatidylserine residues on the wound edge and self-assembles into two-dimensional (2D) arrays [47]. These arrays have been shown to be able to cluster phospholipids, thereby reducing the lateral diffusion of phospholipids [48]. As such, these arrays may help stabilize the wound site until the apparent membrane tension has sufficiently been lowered by other wound-healing mechanisms (reviewed in [35]; see Section 3.1) Indeed, laser ablation experiments performed on murine perivascular cells have shown that the formation of annexin V arrays was necessary for normal wound healing and cell survival [49]. Similar wound stabilization arrays have also been proposed to involve mitsugumin 53 (MG53) oligomers, mini-dysferlinC72 and caveolins [50].
