**5. Tropomyosin family of actin-associated proteins**

Members of the tropomyosin family of actin-associated proteins display a tissue-specific and time-specific expression, while their association with actin filaments impairs a isoform-specific regulation of actin filament dynamics [56]. There are over 40 different isoforms of tropomyosin and many of them have functional relevance to actin filament dynamics: Tm5NM1 and Tm3 increase actin filament resistance to actin depolymerising drugs; TmBr3 increases actin filament sensitivity to actin depolymerising drugs; TmBr3 reduces actin stress fibre formation, while Tm5a and Tm2 inhibit Arp2/3-mediated filament branching in vitro [57]. Tropomyosin proteins assemble as the polymers in the major grove of the polymerised actin filaments and this association has been shown to regulate the molecules that control actin filament turnover [58]. Specific members of the tropomyosin family of actin-associated proteins have yet to be investigated in diabetic wound healing; however, tropomyosin receptor kinase A (TrkA) has been found to be increased in diabetic patients and linked to diabetic nephropathy [59].

**4.1. Filamin-a**

142 Wound Healing - New insights into Ancient Challenges

cutaneous diseases [55].

FLNa acts as a negative regulator of integrin activation by blocking talin binding to the β integrin tail, and subsequent proteolysis and depletion of FLN. Phosphorylation of the β integrin tail dissociates FLN from integrins, hence allowing activation of integrins via talin and other members. FLNa binding with different partners leads to different outcomes in cell adhesion, spreading and migration: association with F-actin leads to formation of orthogonal F-actin networks with unique mechanical and physiological properties; interaction with Migfilin and R-Ras induces and enhances integrin activation respectively; interaction with RalA induces filopodia formation, while interaction with ROCK and Rho GTPases leads to

Human wounds heal through a combination of granulation tissue formation (via production of extracellular matrix and neovascularisation) and wound contraction (via fibroblastmediated contraction). FLNa has been shown to protect fibroblasts against force-induced apoptosis by stabilising cell-matrix contacts [51]. Moreover, fibroblast spreading and adhesion are dependent on FLNa, consistent with its known role in cytoskeletal dynamics [52]. Studies in mice show that FLNa stabilises actin filaments in fibroblasts and mediates wound closure by promoting elastic deformation and maintenance of tension in the collagen matrix [53]. FLNa accumulates at membrane ruffles where it interacts with different binding proteins and regulates fibroblast interactions with their mechanical environment [54]. When FLNa was blocked using short hairpin RNA, fibroblasts were unable to maintain tension in collagen matrices, and they had reduced migration in vitro. In addition, FLNa-deficient fibroblasts were less able to realign collagen matrix fibres in response to tension, and they demonstrated impaired ability to form cell extensions, a deficit reversed with pharmacologic stabilisation of the actin cytoskeleton. When FLNa was deleted conditionally in dermal fibroblasts in a mouse model, full-thickness wounds healed significantly more slowly and was associated with decreased matrix deposition. No side effects or contradictions were observed in these mouse models suggesting that targeted therapies against FLNa may be worth pursuing. As researchers continue to unlock the molecular mechanisms of fibroblast mechanotransduction, novel therapies may be developed to target and manipulate fibroblast behaviour for a wide range of

Members of the tropomyosin family of actin-associated proteins display a tissue-specific and time-specific expression, while their association with actin filaments impairs a isoform-specific regulation of actin filament dynamics [56]. There are over 40 different isoforms of tropomyosin and many of them have functional relevance to actin filament dynamics: Tm5NM1 and Tm3 increase actin filament resistance to actin depolymerising drugs; TmBr3 increases actin filament sensitivity to actin depolymerising drugs; TmBr3 reduces actin stress fibre formation, while Tm5a and Tm2 inhibit Arp2/3-mediated filament branching in vitro [57]. Tropomyosin proteins assemble as the polymers in the major grove of the polymerised actin filaments and

increased actin cytoskeleton remodelling required for cell migration [49].

**5. Tropomyosin family of actin-associated proteins**

High levels of Tm5NM1 expression have been shown to inhibit cell migration and invasion, while loss of Tm5NM1 leads to increased cell motility [57]. Elevated Tm5NM1 expression is associated with inhibition of Src activation [57]; stabilisation of mature focal adhesions [18]; increased myosin II recruitment and actin filament tension [58]; and increased paxillin phosphorylation [18]. These findings suggested that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, including wound healing. The effect of wounding on Tm5NM1 expression has shown that Tm5NM1/2 expression is increased in response to wounding in mice skin and inversely correlates with paxillin phosphorylation and Rac activity regulating lamellapodial protrusions [60]. In addition, the effect of Tm5NM1 and Tm5NM2 isoform knock-down on wound healing using Tm5NM1/2−/− mice showed an accelerated wound healing response with smaller wound area and gape at day 7 post-wounding (**Figure 5**) suggesting a negative role for tropomyosins during wound repair [60]. Increased wound healing was not associated with increased cell proliferation or matrix remodelling but increased cell migration and activation of the paxillin/ Rac signalling, suggesting that tropomyosin isoform expression has an important role in temporal regulation of wound repair [60]. Understanding how different actin remodelling proteins affect wound repair may hold clues for the development of novel therapies aimed at improved wound healing outcomes.

**Figure 5.** Cutaneous wound healing is accelerated at day 7 in Tm5NM1/2−/− mice. Representative H&E-stained transverse sections of full-thickness wounds after 7 days in Tm5NM1/2 and wild-type control mice (lines indicate the edges of the wound area). Adi., adipose tissue; Der., dermis; Ep., epidermis; Pan., panniculus. Bar = 500 μm. Figure adapted from [60].
