*4.1.3.1. Dysferlin-mediated exocytosis*

Muscle fibers are highly mechanically active and endure constant mechanical stresses from movement and exercise, and therefore are prone to stress-related injury. Indeed, their tubular morphology further facilitates the generation of shear stress along the long axis upon eccentric contraction, in which the muscle fiber lengthens while its constituent sarcomeres contract [122]. T-tubules are invaginations of the sarcolemma that run perpendicular to the overall muscle fiber's long axis. These invaginations penetrate deep into the muscle fiber and mediate depolarization of membrane potential required for proper muscle contraction via excitationcontraction coupling. High levels of normal stress exerted on T-tubules upon eccentric contraction may rupture them, thereby severely disrupting the local sarcolemma [123, 124]. As such, muscle cells, especially muscle fibers, evolved potent single-cell repair mechanisms to cope with the constant duress under which they find themselves. For this reason, muscle cells and muscle fibers were instrumental in the study of mechanisms responsible for mammalian somatic single-cell repair.

Dysferlin was initially identified as genetic causes of limb-girdle muscular dystrophy 2B (LGMD2B) [125] and Miyoshi myopathy [126], and dysferlin has since been shown to be ubiquitously expressed with particularly high levels in skeletal muscle, heart and kidney [127]. Its prominence in muscle membrane repair was experimentally demonstrated when dysferlinnull mice were observed to develop progressive limb-girdle muscular dystrophy 2B due to defects in Ca2+-dependent sarcolemma resealing [128]. Dysferlin is a member of the C2 domain-containing ferlin family, which are known regulators of Ca2+-dependent vesicle fusion for auditory neurotransmission [34, 129, 130] and are believe to be functionally similar to synaptotagmin I [131]. Dysferlin's localization at the sarcolemma [132] has led to its research in the context of sarcolemma repair. Indeed, it appears that in muscle cells, dysferlin is at the center of Ca2+-dependent exocytosis following injury, after which intracellular membranes are delivered to the plasma membrane, and ASM released to the outer leaflet [133].

Molecular events involved in dysferlin-mediated exocytosis are a lot more complex than the one involved in synaptotagmin VII/SNAREs-mediated fusions. Indeed, dysferlin has been shown to bind to or be associated with a relatively high number of proteins including MG53 [134, 135], caveolin-3 [134], annexin I [136] and many others (reviewed in [137]). However, a study of human myoblasts by Lek et al. [50] led to the discovery that a calpain-cleaved product of dysferlin played a direct role in the sarcolemma's exocytic repair mechanism. Briefly, calpains activated by injury-induced Ca2+ influx cleave dysferlin, which releases its C-term fragment mini-dysferlinC72 [50, 138, 139]. Following vesicle packaging, mini-dysferlinC72 containing cytoplasmic vesicles are then transported to the wound site. Once localized, minidysferlinC72 interacts with MG53 compartments to form an array, which has been proposed to promote repair by way of wound stabilization (see Section 3.2), as well as promote heterotypic fusion between intracellular vesicles [140] and the sarcolemma [50, 138, 139].

Interestingly, dysferlin has also been shown to associate with AHNAK and may be related to enlargeosome exocytosis. Enlargeosomes are small, AHNAK-positive vesicles resistant to nonionic detergents that undergo endocytosis via a nonacidic route and are supposedly distinct form other conventional vesicular compartments [141]. AHNAK [142] is a very large (≈700 KDa) protein involved in a variety of distinct functions and pathologies (reviewed in [143]). The exact nature and contribution of enlargeosomes to dysferlin-mediated repair is still somewhat ill defined and may vary according to wound severity and cell type. Indeed, their regulated exocytoses have been suggested to add significant amount of membrane components to the injured plasmalemma in neuronal cells [144] and may therefore be involved in either endocytic (see Section 4.1.3.2) or shedding-mediated repair (see Section 4.2.3) [145]. Dysferlin may also modulate plasma membrane repair via its interaction with the AHNAK/ S10010A10/Annexin II complex, which is a known organizer of the actin cytoskeleton and plasma membrane architecture [146].

Hence, contrary to oocytes, dysferlin-mediated exocytosis does not exclusively lead to the formation of a "patch," which also results in diminished in-plane membrane tension and ASM release to the outer leaflet.

### *4.1.3.2. Caveolae-mediated endocytic repair of mechanical wounds*

Exocytosis is insufficient to fully explain the repair of membrane disruptions as lesions from pore-forming proteins are readily removed from the plasma membrane (see Section 4.2.1), which is not explainable by exocytosis alone. Also, the repair of SLO and mechanical lesions has been shown not to depend on exocytosis *per se*, but on the injury-induced release of ASM. Indeed, ASM deficiency, as seen in Niemann-Pick disease (NPD) types A and B [147], is capable of Ca2+-dependent exocytosis but have severely limited Ca2+-dependent endocytosis and shows signs of defective plasma membrane repair, both of which can be rescued by exogenously provided ASM [101, 148]. Similarly, inhibition of ASM by desipramine inhibited both endocytosis and normal plasma membrane repair [101]. This injury-induced endocytosis had previously been described and suggested to be involved in the endocytic degradation of SLO pores and of mechanical disruptions [149]. The same study identified the endosomes involved to be Ca2+- and cholesterol dependent [149], but did not offer any mechanistic insight into their formation. As previously stated, caveolae are lipid-raft-rich whose formation is dependent on cholesterol, PIP2 and PS (see Section 3.1), and are known to be facilitated by the transient formation of ceramide on lipid rafts [150, 151]. Once released to extracellular fluid, ASM cleaves the phosphorylcholine heads of sphingomyelin leaflets on the membrane surface to generate ceramide sphingolipids [152]. The resulting ceramide-enriched domains of the phospholipid bilayer are more prone to membrane invaginations due to them encompassing a smaller molecular area relative to other membrane lipids [152], promoting caveolae's endocytic function [153–155]. Similarly, caveolin-3 deficiency causes muscle degeneration in mice [134], which mirrors the limb-girdle muscular dystrophy 1C (LGMD1C) phenotype in humans [156]. As such, exocytosis of lysosomes and dysferlin vesicles after plasma membrane injury may not heal the membrane directly, but rather facilitate membrane resealing by encouraging caveolae formation. Indeed, upon heterotypic fusion with the membrane, ASM is released to the outer surface of the membrane, which potentiates the formation of ceramide-rich platforms that have been shown to trigger invagination of the plasmalemma [157] and formation of caveolae-derived endosomes (reviewed [158]). Indeed, transmission electron microscopy (TEM) has shown that caveolae were found to be concentrated next to mechanical disruptions of muscle cells [153] and assemble into a single, large merged caveolae-like structure around the large wounds generated in primary muscle fibers [153]. As Corrotte et al. correctly pointed out, these very large endocytic vesicles and invaginations may have initially been identified as related to the exocytic "patch" that was initially proposed to cover and eventually heal wounds in muscle cells (see Section 4.1.3.1). Alternatively, Corrotte et al. [153] proposed an endocytic-mediated model of plasma membrane repair. Briefly, large caveolae-like invaginations are formed as a consequence of a combination of the lower in-plane tension provided by the exocytosis of lysosomes, dysferlin-positive vesicles, changes in plasma membrane shape that follows release of ASM, and the presence of proteins such as dysferlin and caveolins. The growth and eventual fusion of those caveolae-like invaginations provides a "constriction force" that promotes plasmalemma resealing [153].

nonionic detergents that undergo endocytosis via a nonacidic route and are supposedly distinct form other conventional vesicular compartments [141]. AHNAK [142] is a very large (≈700 KDa) protein involved in a variety of distinct functions and pathologies (reviewed in [143]). The exact nature and contribution of enlargeosomes to dysferlin-mediated repair is still somewhat ill defined and may vary according to wound severity and cell type. Indeed, their regulated exocytoses have been suggested to add significant amount of membrane components to the injured plasmalemma in neuronal cells [144] and may therefore be involved in either endocytic (see Section 4.1.3.2) or shedding-mediated repair (see Section 4.2.3) [145]. Dysferlin may also modulate plasma membrane repair via its interaction with the AHNAK/ S10010A10/Annexin II complex, which is a known organizer of the actin cytoskeleton and

Hence, contrary to oocytes, dysferlin-mediated exocytosis does not exclusively lead to the formation of a "patch," which also results in diminished in-plane membrane tension and ASM

Exocytosis is insufficient to fully explain the repair of membrane disruptions as lesions from pore-forming proteins are readily removed from the plasma membrane (see Section 4.2.1), which is not explainable by exocytosis alone. Also, the repair of SLO and mechanical lesions has been shown not to depend on exocytosis *per se*, but on the injury-induced release of ASM. Indeed, ASM deficiency, as seen in Niemann-Pick disease (NPD) types A and B [147], is capable of Ca2+-dependent exocytosis but have severely limited Ca2+-dependent endocytosis and shows signs of defective plasma membrane repair, both of which can be rescued by exogenously provided ASM [101, 148]. Similarly, inhibition of ASM by desipramine inhibited both endocytosis and normal plasma membrane repair [101]. This injury-induced endocytosis had previously been described and suggested to be involved in the endocytic degradation of SLO pores and of mechanical disruptions [149]. The same study identified the endosomes involved to be Ca2+- and cholesterol dependent [149], but did not offer any mechanistic insight into their formation. As previously stated, caveolae are lipid-raft-rich whose formation is dependent on cholesterol, PIP2 and PS (see Section 3.1), and are known to be facilitated by the transient formation of ceramide on lipid rafts [150, 151]. Once released to extracellular fluid, ASM cleaves the phosphorylcholine heads of sphingomyelin leaflets on the membrane surface to generate ceramide sphingolipids [152]. The resulting ceramide-enriched domains of the phospholipid bilayer are more prone to membrane invaginations due to them encompassing a smaller molecular area relative to other membrane lipids [152], promoting caveolae's endocytic function [153–155]. Similarly, caveolin-3 deficiency causes muscle degeneration in mice [134], which mirrors the limb-girdle muscular dystrophy 1C (LGMD1C) phenotype in humans [156]. As such, exocytosis of lysosomes and dysferlin vesicles after plasma membrane injury may not heal the membrane directly, but rather facilitate membrane resealing by encouraging caveolae formation. Indeed, upon heterotypic fusion with the membrane, ASM is released to the outer surface of the membrane, which potentiates the formation of ceramide-rich platforms that have been shown to trigger invagination of the plasmalemma [157] and formation of

plasma membrane architecture [146].

206 Wound Healing - New insights into Ancient Challenges

*4.1.3.2. Caveolae-mediated endocytic repair of mechanical wounds*

release to the outer leaflet.

Endocytosis leads to a decrease in total plasma membrane surface area, increasing in-plane membrane tension and providing the force necessary for the mechanical wound removal. It is, however, important to consider that exocytosis of ASM lysosomes always precedes caveolae-mediated endocytosis. The corresponding in-plane membrane tension increases likely readjusts overall apparent membrane tension back to the cell's pre-injury levels, as type-I alveolar epithelial cells are known to remediate to hypertonic shock by increased caveolaemediated endocytosis [159]. In fact, endocytic repair seems not to be as muscle specific as it was once thought since alveolar cell repair has been suggested to be linked to MG53 and caveolin-1 [160, 161].
