**2. Actin dynamics during wound healing**

Actin-based cell motility relies on the balanced activity of specific actin-binding proteins that drive the dynamics of the actin system and govern its special organisation [6]. A number of different structural and dynamic aspects of cell behaviour are dependent on the actin cytoskeleton, including cell morphology, polarity, adhesion complex formation, vesicle trafficking and phagocytosis, cytokinesis and movement [7]. Actin microfilaments are the smallest components of the actin cytoskeletal network and play a role in cellular motility, structure and division [6]. Two types of actin microfilaments have been categorised; individual non-polymerised globular actin subunits termed G-actin and long filamentous polymerised fibres termed F-actin assembled from individual G-actin subunits. Microfilament actin (F-actin) exists in equilibrium with a soluble monomeric actin (G-actin) and this balance is often shifted in response to changes in cellular environment, cellular migration, adhesion and wound repair [8]. During wound healing, activation of neutrophils during the inflam‐ matory phase of wound repair induces changes in cell shape, migration, degranulation and phagocytic responses, all of which require cytoskeletal restructuring. In addition, the rees‐ tablishment of the skin barrier function as well as endothelial vessel integrity in wounds is dependent on actin cytoskeleton integrity [9]. Microtubules and intermediate filaments are larger structures of the cytoskeleton composed of α and β tubulin dimers which function in both cellular movement and division [6]. Intermediate filaments are involved in the forma‐ tion of adhesion complexes namely hemidesmosomes, desmosomes and focal adhesions and directly interact with proteins of the extracellular matrix [10]. Key roles of the inter‐ mediate filaments include signal transduction, cytoskeletal crosstalk between the organelles in the cytoplasm and organisation of the cytoplasm [11].

is also involved in modulating cell signalling, growth, differentiation and gene expression, while components of the actin cytoskeleton further work in synergy to provide stronger cell

Cutaneous wound repair is a dynamic process triggered in response to tissue injury, which aims to restore the skin barrier function, and involves a sequence of events including acute inflammation, reepithelialisation, collagen deposition and contraction and remodelling [3]. Common to all tissue repair processes is the migration of cells into the wound space including fibroblasts, epithelial cells and endothelial cells. It is the active assembly and disassembly of the filamentous actin and reorganisation of its networks that underpins the important cell

Changes in the distribution of actin-associated proteins during epidermal wound healing in vivo were first reported in 1992 [4]. Filamentous actin was found in all the living epidermal layers before, after and during wound healing while different actin-associated proteins, namely talin, filamin and gelsolin, showed a reduced expression at the leading edge of migrating epidermis, which returned to normal levels once the epidermis has reformed [4]. The precise orchestration of actin polymers into filaments and their interactions with various proteins regulating actin remodelling, stability, branching and bundling is what underpins cellular migration and outcomes of wound healing. Central to the ability of fibroblasts and keratinocytes to move into the wounded area is a dynamic and responsive actin cytoskeleton and the molecules that regulate actin filament dynamics and change the rate of cell migration can also alter the rate of wound healing [5]. Understanding the role of the actin cytoskeleton in cellular functions vital for tissue repair and regeneration and how different regulators of the actin cytoskeleton control this intricate balance between actin polymerisation and disassembly will be critical for the development of novel therapeutic approaches. New therapies that can regulate the actin cytoskeleton could lead to improved wound healing outcomes. Here, we will focus on describing the role of different actin cytoskeleton regulators and how they are able to modulate the cytoskeleton and influence different stages of

Actin-based cell motility relies on the balanced activity of specific actin-binding proteins that drive the dynamics of the actin system and govern its special organisation [6]. A number of different structural and dynamic aspects of cell behaviour are dependent on the actin cytoskeleton, including cell morphology, polarity, adhesion complex formation, vesicle trafficking and phagocytosis, cytokinesis and movement [7]. Actin microfilaments are the smallest components of the actin cytoskeletal network and play a role in cellular motility, structure and division [6]. Two types of actin microfilaments have been categorised; individual non-polymerised globular actin subunits termed G-actin and long filamentous polymerised fibres termed F-actin assembled from individual G-actin subunits. Microfilament actin (F-actin) exists in equilibrium with a soluble monomeric actin (G-actin) and this balance is

stability during stress [1, 2].

134 Wound Healing - New insights into Ancient Challenges

wound healing.

processes, which occur during wound healing.

**2. Actin dynamics during wound healing**

Stress fibres are also a component of the actin cytoskeleton network allowing a cell to modulate its responses to tissue injury. Mammalian cells contain three types of stress fibres: ventral stress fibres attached to focal adhesions at both ends, dorsal stress fibres attached to focal adhesions at one end, and transverse arcs which are the acto‐myosin bundles that do not attach to focal adhesions directly [12]. The major role of stress fibres is to maintain a balance between contraction and adhesion. This balance results in stable actin bundles, which maintain a constant length under tension, especially in ventral stress fibres attached to the extracellular matrix on both sides [13].

Changes in cell shape, adhesion and migration properties are all regulated by the continuous remodelling of the actin cytoskeleton. Cell motility is powered by controlled assembly and disassembly of the actin cytoskeleton, and the migration speed is dependent on the membrane tension created by the coalescence of the actin filaments growing against the tense membrane [14]. In order to migrate in response to extracellular signals, cells first assemble actin at the cell front driving the extension of membrane protrusions called lamellipodia and filopodia [15]. At the leading edge of the cell, adhesions are formed with the extracellular matrix, hence anchoring the protrusions to move the cell body. The combination of the acto‐myosin con‐ tractibility and disassembly of the adhesion structures at the rear of the cell allows the cell body to move forward [8]. Lamellipodia, filopodia and membrane ruffles are components of the actin cytoskeleton involved in both cell motility and cell‐matrix adhesions [16]. Lamelli‐ podia consist of a network of branched actin filaments that produce the force for cell protru‐ sions at the leading edge. The assembly of actin‐based projections is regulated by GTPases of the Rho family, which link the surface receptors to the organisation of the actin cytoskeleton. While Rho GTPase is instrumental in formation of stress fibre and focal adhesion formation, Rac1 and Cdc42 signal the formation of lamellipodia and filopodia, respectively.

Filapodia are thin cellular processes consisting of long parallel actin filaments arranged into tight bundles. Membrane ruffling is characterised by the dynamic movement of the membrane protrusions, consisting of lamellipodia and filopodia, in response to the extracellular signals. Away from the leading edge of the cell at the site of slow actin turnover, lamellas are formed and are characterised by proteins involved in the movement of stress fibres, namely tropo‐ myosin and myosin II [8]. Initial integrin mediated cell‐matrix adhesions, termed focal complexes, develop underneath lamellipodia and are driven by actin polymerisation. These are highly dynamic structures that exist for a limited time. A proportion of the stable focal complexes develop into elongated focal adhesions, which are associated with contractile stress fibres [17]. A vital function of focal adhesions is the anchoring of polymerised actin filament stress fibres into bundles, which provide contractile force required for effective translocation of a cell body during cellular migration [18]. Different components of the actin cytoskeleton of the moving cell and adhesion sites formed in response to GTPase signalling in fibroblasts are shown in **Figure 1**. The main changes in the actin cytoskeleton during wound healing include lamellipodia remodelling during keratinocyte migration and wound reepithelialisation, infiltration of inflammatory cells and migration of fibroblasts required for the deposition and remodelling of the extracellular matrix and dermal wound contraction [19, 20].

**Figure 1.** Actin cytoskeleton of the moving cell. (A) Arrangement of the actin cytoskeleton in a moving cell A lamellipodia, B filopodium, C focal adhesion, D lamella, E focal complex. (B) Formation of different actin cytoskeleton components in response to GTPase signalling in fibroblasts. Actin filaments visualised with phalloidin staining in A, C, E and G and adhesion complexes visualised with an anti-vinculin antibody in B, D, F and H. Quiescent fibroblasts in A and B show few organised actin filaments or adhesion complexes. In response to Rho stress fibre formation C and adhesion complex formation D is evident. Microinjection of Rac induces lamellipodia E and associated focal adhesion complexes, while microinjection of Cdc42 induces filopodia formation G and associated adhesion complexes H. Figure adapted from [8, 25].

In resting cells, there is little actin turnover, and fast-growing actin ends are blocked, with large pools of actin monomers in a complex with polymerising-inhibiting or sequestering proteins. In response to wounding, a local increase in actin polymerisation is initiated by uncapping the actin ends and by severing existing filaments leading to *de novo* polymerisation. The barbed ends of the actin filament are the hotspots for the majority of biochemical reactions that control filament assembly and a number of actin remodelling, capping, severing and sequestering proteins modulate their affinity for barbed ends in a spatial and temporal manner [21]. Some actin remodelling proteins also affect the actin filament barbed ends by indirect activity and control of the flux of actin monomers available at the barded end [22]. Signal transduction networks that translate environmental signals into intracellular changes govern actin dynamics and interplay between extracellular environment and cell motility. Many actin-binding proteins accumulate at sites of actin-rich lamella and have been shown to regulate actin dynamics in motile keratinocytes [23]. Focal adhesion formation in fibroblasts is a complex process initiated by the ligation and clustering of the integrin subunits and signalling via RhoGTPases, which influence both actomyosin contractibility and actin stress fibre formation [24].

complexes, develop underneath lamellipodia and are driven by actin polymerisation. These are highly dynamic structures that exist for a limited time. A proportion of the stable focal complexes develop into elongated focal adhesions, which are associated with contractile stress fibres [17]. A vital function of focal adhesions is the anchoring of polymerised actin filament stress fibres into bundles, which provide contractile force required for effective translocation of a cell body during cellular migration [18]. Different components of the actin cytoskeleton of the moving cell and adhesion sites formed in response to GTPase signalling in fibroblasts are shown in **Figure 1**. The main changes in the actin cytoskeleton during wound healing include lamellipodia remodelling during keratinocyte migration and wound reepithelialisation, infiltration of inflammatory cells and migration of fibroblasts required for the deposition and

**Figure 1.** Actin cytoskeleton of the moving cell. (A) Arrangement of the actin cytoskeleton in a moving cell A lamellipodia, B filopodium, C focal adhesion, D lamella, E focal complex. (B) Formation of different actin cytoskeleton components in response to GTPase signalling in fibroblasts. Actin filaments visualised with phalloidin staining in A, C, E and G and adhesion complexes visualised with an anti-vinculin antibody in B, D, F and H. Quiescent fibroblasts in A and B show few organised actin filaments or adhesion complexes. In response to Rho stress fibre formation C and adhesion complex formation D is evident. Microinjection of Rac induces lamellipodia E and associated focal adhesion complexes, while microinjection of Cdc42 induces filopodia formation G and associated adhesion complexes H. Figure

In resting cells, there is little actin turnover, and fast-growing actin ends are blocked, with large pools of actin monomers in a complex with polymerising-inhibiting or sequestering proteins. In response to wounding, a local increase in actin polymerisation is initiated by uncapping the actin ends and by severing existing filaments leading to *de novo* polymerisation. The barbed ends of the actin filament are the hotspots for the majority of biochemical reactions that control filament assembly and a number of actin remodelling, capping, severing and sequestering proteins modulate their affinity for barbed ends in a spatial and temporal manner [21]. Some actin remodelling proteins also affect the actin filament barbed ends by indirect activity and control of the flux of actin monomers available at the barded end [22]. Signal transduction networks that translate environmental signals into intracellular changes govern actin dynamics and interplay between extracellular environment and cell motility. Many actin-binding proteins accumulate at sites of actin-rich lamella and have been shown to regulate actin dynamics in motile keratinocytes [23]. Focal adhesion formation in fibroblasts is a complex process initiated by the ligation and clustering of the integrin subunits and signalling via

adapted from [8, 25].

remodelling of the extracellular matrix and dermal wound contraction [19, 20].

136 Wound Healing - New insights into Ancient Challenges

For cellular migration, dynamic rearrangements of the actin cytoskeleton occur to form protrusive structures and generate intracellular forces required for cell movement. The actinbased motility is best described in four steps: polarisation, protrusion of lamellipodia, formation of attachment sites and retraction of cell rear end [6]. Fibroblast locomotion during wound healing is the result of series of coordinated cellular events and main motor protein involved in mediating formation of lamellipodia of migrating cells is Myosin I. However, during wound healing, Myosin II, motor protein, is involved in the contraction of transverse actin fibres during lamellar contractile phase of wound healing [26]. In addition, release of Myosin II contractibility accelerates the healing of large wounds in long term by mobilisation of large cell sheet or rows of cells behind the leading edge [27].

**Figure 2.** The role of actin cytoskeleton in regulating myofibroblast function. Actin cytoskeleton involvement in bidirectional signalling augmenting extracellular matrix organisation, focal adhesion turnover and contraction as well as transcriptional regulation of proteins instrumental in these processes vital for outcomes of wound repair. Figure adapted from [24].

Myofibroblasts are modified fibroblasts characterised by the presence of the contractile apparatus and formation of robust stress fibres. These cells are involved in the contraction and remodelling of the extracellular matrix but are also found in aberrant tissue remodelling in fibrotic disorders. The actin cytoskeleton regulates several mechanical functions during myofibroblast differentiation including focal adhesion formation, contraction and matrix remodelling and simultaneously regulates transcription of genes involved in the same mechanical functions and therefore plays an important role in amplifying the signal leading to myofibroblast differentiation. The bidirectional signalling between matrix stiffness, focal adhesion augmentation and stress fibre formation during actin cytoskeletal regulation of myofibroblast function is illustrated in **Figure 2** [28].

### **2.1. Scar-free foetal and adult wound healing**

Whereas adult wound keratinocytes crawl forwards over the exposed substratum closing the deficit, a wound in embryonic epidermis is closed by contraction of an actin purse string. Blocking the assembly of this actin cable in chick and mouse embryos by drugs or by inactivation of small GTPase Rho severely hinders the reepithelialisation process [29]. Foetal wounds reepithelialise quickly via contraction of actin-myosin fibres in a "purse-string" like manner drawing the edges of the wound together. This is facilitated by the rapid polymerisation of the F-actin some five to six cells back from wound edge and is anchored by the Ecadherin at the leading edge to facilitate coordinated movement [30]. Foetal wound fibroblasts do not express alpha smooth muscle actin, suggesting that they do not change their phenotype into contractile myofibroblasts observed in adult wounds and these differences may account for differences in repair outcomes in foetal vs adults wound tissue [30]. Changes in the expression profile of proteins associated with actin cytoskeleton are indicative of the switch between scar-free regeneration and scar forming repair. Wounding has a differential effect on cytoskeletal proteins including gelsolin and paxillin associated with actin dynamics both in foetal and adult skin wounds [19, 31]. Interestingly, wounding also has an effect on the expression of filamentous F-actin. While "scar-free" foetal wounds have predominantly epidermal expression of F-actin, the "scar forming" adult wounds have predominantly dermal F-actin expression and this developmental switch in actin expression might be important in foetal wound contraction and "scar-free" wound healing [32]. The importance of the actin cytoskeleton in healing of foetal wounds was demonstrated at embryonic day E17 by the addition of cytochalasin-B, an inhibitor of actin polymerisation, which completely prevented epithelial wound closure with no actin cable structures evident while at embryonic day E19, dermal actin filaments formed spherical structures around the wound margin but did not affect already limited wound repair response (**Figure 3**) [19].

**Figure 3.** Effect of inhibiting actin polymerisation and protein proliferation on actin cable formation in E17 foetal wounds. Phalloidin-FITC binding to actin in E17 and E19 skins foetal skin treated with 10 μg cytochalasin-B per ml (A and B, respectively). Phalloidin-FITC binding to actin in E17 foetal skin treated with 2 mM hydroxyurea (C). Scale bar = 50 μm in (C) and applies to all images. Figure adapted from [19] and modified.

### **3. FERM superfamily of proteins**

The FERM domain (F for 4.1protein, E for ezrin, R for radixin and M for moesin) is a widespread domain found in many cytoskeletal associated proteins at the interface between the plasma membrane and the actin cytoskeleton. The function of FERM domain is to localise the proteins to the plasma membrane, and therefore, members of the FERM superfamily of proteins mediate the linkage between the actin cytoskeleton and cell membrane and are characterised by the presence of the conserved FERM domain at the N-terminus and often an actin binding domain at the C-terminus. FERM proteins are involved in cellular motility and membranecytoskeletal interactions and play roles in promotion of cancer and wound healing [33]. The main members of this family include protein 4.1R, ezrin, radixin and moesin, often referred to as ERM proteins. Ezrin is a component of the microvilli of the plasma membrane, meosin is involved in binding major cytoskeletal structures to the plasma membrane, while radixin is involved in the binding of the barbed end of actin filaments to the plasma membrane [21, 34]. Earlier studies have shown that ezrin, radixin and meosin colocalise with F-actin in the endothelial cells in vitro and in vivo and play a role in formation of focal F-actin branching points, while their interaction with phosphorylated protein kinase C (PKC) has been shown to be important during wound repair [35]. Inhibition of PKC activity results in delayed wound repair, reduced association with ERM proteins and reduced F-actin branching points. In addition, phosphorylation of ERM proteins by PCK improved in vitro wound healing of cancer cells [36]. Furthermore, in vivo studies examining the healing of hepatic injury in meosin knockdown mice showed reduced inflammatory infiltrate, fibrosis and collagen deposition at the wound margins of these mice compared with control animals [37].

string. Blocking the assembly of this actin cable in chick and mouse embryos by drugs or by inactivation of small GTPase Rho severely hinders the reepithelialisation process [29]. Foetal wounds reepithelialise quickly via contraction of actin-myosin fibres in a "purse-string" like manner drawing the edges of the wound together. This is facilitated by the rapid polymerisation of the F-actin some five to six cells back from wound edge and is anchored by the Ecadherin at the leading edge to facilitate coordinated movement [30]. Foetal wound fibroblasts do not express alpha smooth muscle actin, suggesting that they do not change their phenotype into contractile myofibroblasts observed in adult wounds and these differences may account for differences in repair outcomes in foetal vs adults wound tissue [30]. Changes in the expression profile of proteins associated with actin cytoskeleton are indicative of the switch between scar-free regeneration and scar forming repair. Wounding has a differential effect on cytoskeletal proteins including gelsolin and paxillin associated with actin dynamics both in foetal and adult skin wounds [19, 31]. Interestingly, wounding also has an effect on the expression of filamentous F-actin. While "scar-free" foetal wounds have predominantly epidermal expression of F-actin, the "scar forming" adult wounds have predominantly dermal F-actin expression and this developmental switch in actin expression might be important in foetal wound contraction and "scar-free" wound healing [32]. The importance of the actin cytoskeleton in healing of foetal wounds was demonstrated at embryonic day E17 by the addition of cytochalasin-B, an inhibitor of actin polymerisation, which completely prevented epithelial wound closure with no actin cable structures evident while at embryonic day E19, dermal actin filaments formed spherical structures around the wound margin but did not affect already limited wound repair response (**Figure 3**) [19].

**Figure 3.** Effect of inhibiting actin polymerisation and protein proliferation on actin cable formation in E17 foetal wounds. Phalloidin-FITC binding to actin in E17 and E19 skins foetal skin treated with 10 μg cytochalasin-B per ml (A and B, respectively). Phalloidin-FITC binding to actin in E17 foetal skin treated with 2 mM hydroxyurea (C). Scale

The FERM domain (F for 4.1protein, E for ezrin, R for radixin and M for moesin) is a widespread domain found in many cytoskeletal associated proteins at the interface between the plasma membrane and the actin cytoskeleton. The function of FERM domain is to localise the proteins

bar = 50 μm in (C) and applies to all images. Figure adapted from [19] and modified.

**3. FERM superfamily of proteins**

138 Wound Healing - New insights into Ancient Challenges

The role of the FERM protein superfamily during wound healing has also been demonstrated in 4.1R knockout mice (4.1R2013/ 2013) and their cultured keratinocytes. Protein 4.1R is present in the cytoplasm and the leading edge of the moving cell [34]. Absence of 4.1R protein leads to reduced adhesion, spreading and migration of keratinocytes. In addition, diminished focal adhesion complexes and reduced integrin Beta-1 expression were directly linked to absence of 4.1R protein in vitro. Using its FERM domain, 4.1R protein was shown to interact with the Ras GTPase-activating-like protein 1, a scaffolding protein that binds and cross-links actin filaments, allowing migration to take place [34]. In addition, ezrin/radixin/moesin proteins have been shown to be involved in the development of diabetes including the secretion and utilisation of insulin and may contribute to the pathogenesis and progression of diabetic angiopathy, nephropathy and cardiomyopathy [38]; all of which have been implicated in the development of diabetic ulcers. These proteins may be novel targets for therapeutic interventions aimed at preventing diabetic complications; however, further research is required to elucidate their exact mechanisms before they can be developed for specific treatments.

The FERM superfamily of proteins consists of over 30 proteins including the Kindlin family of focal adhesion proteins. Kindlin-1 and Kindlin-2 have been implicated in integrin signalling and focal adhesion turnover, while deletion of the Kindlin-1 is associated with a congenital skin disease—Kindler Syndrome, where patients experience skin atrophy, blister formation and impaired wound healing [39–42]. Kindlin-2 is an important regulator of focal adhesion stabilisation and maturation of focal adhesions and stress fibres in myofibroblasts. In addition, the upregulation of Kindlin-2 observed in myofibroblasts during wound healing suggests a role for Kindlin-2 in skin fibroblasts and tissue regeneration [41]. More recently, talin and Ehm2 have been added to the FERM superfamily both of which have roles in wound repair [21, 34].

#### **3.1. Talin**

Talin, a member of the FERM family of proteins, is concentrated in regions of cell-substrate and cell-cell contacts. Using its FERM domain, talin acts as a "hyper-activator" of integrin receptors linking the cytoplasmic tail of integrin receptors to the actin cytoskeleton and increasing the affinity of the integrin extracellular domain to the extracellular matrix, hence regulating cell adhesion-dependent processes including tissue remodelling [43]. Talin knockout results in abnormal cellular migration and early embryonic lethality [8]. Integrin adhesion receptors connect the extracellular matrix to the actin cytoskeleton and serve as bidirectional mechanotransducers during wound healing mediating actin cytoskeletal remodelling in response to stiffening of the extracellular matrix [44]. The inside out-signalling of integrin receptors regulates the ligand binding affinity of the cell surface receptors in response to changes in environmental factors important for cell survival, including tissue injury [45]. Cytoplasmic talin is activated in response to phosphatidylinositol 4,5-biphosphate (PIP2) binding which also terminates the auto-inhibition of talin through the talin head-rod binding. Once activated, the talin subdomain interacts with the β integrin tails, forms the talin specific

**Figure 4.** Talin activation of integrin receptor subunits. (A) PIP2 binding to the cytoplasmic talin activates the talin protein by ending the auto-inhibitory interaction with the rod domain. (B) Talin subdomain engages with the membrane proximal NPxY motif in the β integrin cytoplasmic tail. (C) Talin-specific loop structure forms with binding to the MP helical region of the integrin cytoplasmic chain, hence disrupting the connection between α/β subunit integrin cytoplasmic tails. Pulling forces at the β integrin tail reorient the transmembrane domains, hence disrupting the packing of the α/β transmembrane domains. Figure adapted from [46].

loop structure and disrupts the connection between the cytoplasmic tails linking the integrin receptors and the actin cytoskeleton [46]. This model of integrin activation by talin is an example of how cytoplasmic proteins can regulate activation state of integrin receptors and can transduce the biochemical signals into an array of cellular signalling transduction pathways, a crucial function for cellular adhesion, migration, angiogenesis, extracellular matrix assembly and wound remodelling [47]. **Figure 4** illustrates a schematic model of talin activation of integrin subunits; however, this process is likely to be more complex and involves spatial activation and interaction of different proteins involved in actin dynamics. Recent findings have shown that talin associates with the actin remodelling protein Flightless I (Flii) in wounded keratinocytes and this interaction may contribute to Flii regulation of adhesiondependent signalling pathways during wound repair [48].
