Cell migration is a fundamental process responsible for numerous physiological and physiopathological conditions such as inflammation, embryogenesis and cancer. This central aspect of cell biology has seen quantum leaps in our understanding of the coordinated regulations, both spatial and temporal of numerous cytoskeletal proteins and their orchestrations. At the molecular level, this dynamic cellular process can be naively summarised as an engineered cycle composed of three distinct phases of (1) formation of cellular protrusion to initiate contact followed by (2) the adhesion with the external environment/cell-extracellular established connection and (3) the actomyosin force generation to consequently remodel the cytoskeleton. A prominent factor that regulates cellular motility is S100A4, a protein that has received constant attention for its significant role in cellular migration. Consequently, and in order to focus further the impact of this work, the present chapter aims to review some of the actomyosin proteins/complexes that have been demonstrated to be crucial players of the cyclic migration process but are also S100A4 interactors. In doing so, this chapter aims to capture a picture of how expression of this small, calcium-binding protein may, in essence, remodel at different levels the actin organisation and fulfil the motility engineered cycle of protrusion, attachments and contractions.
Cellular motility has been an essential cellular phenomenon throughout phylogeny that has allowed organisms to survive, adapt and prosper in different environments. It is engrained in the chemoattraction and nutrient-seeking mechanisms in protozoa such as
Other than these physiological conditions, cellular motility is essential in regulating some of the physiopathological steps seen in disease. As example, it is well-documented that cellular migration is one of the prominent factors involved in the later stages of carcinogenesis and the subsequent phases of metastasis [9–11]. Cancer cell dissemination is clearly dependent upon the ability of migratory tumour cells to evade away from their initial niche, leading to the colonisation and formation of distant secondary lesions in the body .
At the molecular level, cell migration requires the coordinated regulations, both spatial and temporal of numerous cytoskeletal proteins, to orchestrate the dynamic cellular processes needed for cells to acquire movement. In this context, the actin cytoskeleton and the closely linked myosin network play essential functions [13, 14]. The process of cellular motility can be summarised as an engineered cycle composed of three distinct phases which are, (1) formation of cellular protrusion in the forms of lammelipodia and fillopodia to initiate contact and adhesion with the external environment, (2) regulation of cell-extracellular matrix established connections, usually integrin-dependent, and (3) force generation by the actomyosin network which will control both the structure and organisation of the motile architecture . I provide here a brief overview of some of the different elements and protein complexes that are regulated during this migratory cycle, focusing primarily on specific components of the actomyosin complexes.
A group of low-molecular weight polypeptides that has been demonstrated to have key functions in remodelling the overall actin cytoskeletal network is the S100 protein family . Composed of approximately 25 members, the presence of the majority of these in different cellular systems, both in vivo and in vitro has been associated with significant changes in cellular migration. One of the most prominent members of this family to have been linked to regulate cellular motility is S100A4, a protein that has received constant attention for its significant role in promoting cancer metastasis [16–18]. Consequently, and in order to emphasis the impact of this work and strengthened its delivery, I have concentrated our attentions on actomyosin proteins/complexes that have both been demonstrated to be crucial players of the migration process but also S100A4 interactors. In doing so, this chapter aims to capture a picture of how expression of this small, calcium-binding protein may in essence remodel at different levels the actin organisation and fulfil the motility engineered cycle of protrusion, attachments and contractions.
2. The actomyosin machinery in cellular migration
Motility can be seen as a lone activity where a single cell may migrate (also known as amoeboid or mesenchymal migration [19, 20]) or is referred to as collective, if this effort is the result of concerted effort undertaken by numerous cells, either in sheet or clusters . Equally important is the cell physiognomy that will be regulated in the process. Mesenchymal motility as seen during fibroblast migration leads to cellular characteristic of a more elongated spindle-like shape. In this type of migration, an actin-rich leading edge can be observed, where extension of the front leading edge is driven by actin polymerisation . In amoeboid migration, cells adopt a more rounded morphology and rely on the contraction-based membrane blebbing and enriched levels of myosin II at the cell rear . Both of these migratory processes have been shown to play important roles in both physiological and pathological events.
The complexity of the different types of cell migration that can be used is mirrored by the number of different molecular pathways that are available to orchestrate these processes. Among them, however, the remodelling of the actin cytoskeleton and its organisation stands as an irreplaceable circuitry which is undisputably common to all. At the molecular level, this network is considered to provide the platform where physical forces will be exerted. Pushing forces generated by the assembly of filamentous actin (F-actin) will encourage the formation of cellular protrusions, such as filopodia, lamellipodia, blebbing and the most recently characterised invadopodia [23–25]. These changes in actin polymerisation and their dynamics will be directly responsible for reshaping and remodelling the underling plasma membranes.
2.1. Cellular protrusions and regulators
In the early stages of assembly, also known as nucleation, actin protomers aggregate in an energetically unfavourable process to form a dimer that is more likely to dissociate. Addition of another subunit stabilises the complex and represents the nucleus, a state where actin polymerisation is now more favourable than dissociation (Figure 1). The association of monomers into a trimeric structure is seen as the rate limiting step of the whole polymerisation process as it is reversible where monomers can easily dissociate [28–30]. It is during the stage of nucleation that addition of further actin subunits is supported at both ends. Once the nucleus and newly added monomers have been locked into position by conformational changes, the process of elongation begins and the addition of actin molecules at the barbed end of the filament can be seen, resulting in the formation of structural polarised complexes (Figure 1). Whilst G-actin subunits can self-assemble, this process only occurs if the concentration of actin exceeds a critical concentration.
Within cells, a growing number of binding partners, or actin-binding proteins, will act both antagonistically and agonistically to regulate the polymerisation process. Some factors will act as nucleators, such as formins and Arp2/3, facilitating the process through providing a scaffold structure which encourages de novo assembly. Others will regulate the overall structure of filaments through their remodelling in larger structures. Examples provided here will control the cross-linked state of actin filaments through the involvement of bundling regulators such as the tropomyosins and to an extent myosins. Involvements of all these factors, as well as many others that are too numerous to be listed, here, will be responsible for the remodelling of the actin cytoskeleton into different substructures seen during cell migration (Figure 2).
When grown in a 2D environment, cells will encourage the formation of differential planar filamentous actin, in the form of filipodia/microvilli or sheet-like structures referred to as lamellipodia (Figure 2, [31, 32]). Whilst the former act as sensory organelles that enable cells to probe their local environment, through the formation of thin extensions that are mainly made of long, unbranched bundles, the latter is viewed as the main driving force for locomotion, through the organisation of short branched actin networks (Figure 2). In both instances, however, regulation of F-actin polymerisation, especially at their barbed end is essential, in order to control their elongation in the direction of the plasma membrane and is thought to require nucleation-promoting factors where both formins and Arp2/3 have been shown to play key functions (Figure 2, [31, 33]).
Taken all together, actin polymerization at the leading edge is a vital process for cellular migration, through the orchestrations of events that will ultimately lead to different cellular protrusion events. In this section, different actin polymerization factors and their functions (Arp2/3 and Formin) were briefly explored. One should remember that this is only a preferential view in regards to their potential involvements through a S100A4-dependent process and that numerous other regulators not mentioned here play equally vital roles in the process of actin remodelling and cellular migration.
Away from the leading edge and the protrusions of the lamellipodia and filopodia, the array of filamentous actin is seen to exist as more bundling rather than the branched sheets reported previously, mainly due to the interaction of different actin-binding proteins. This contractile network is seen as a unique structural complex, spatially posterior to the lamellipodium, and is referred to as the lamellum .
2.2. Lamellum and cellular contractions
In the spatial arrangement of the lamellum, filaments are organised in different structures, known as stress, dorsal and ventral fibres; they are the result of interaction of the actin filaments with different partners (Figure 2). It is in this context and primarily through the control of the tropomyosin and myosins that contractile forces are exerted to manipulate their overall organisation. Generation of such tensile forces is provided by the myosin network, mainly non-muscle myosin II (NMMII) which is responsible for the majority of the morphological and architectural reorganisations that promote cell movement.
The overall organisation of the actin cytoskeleton can also be dictated by actin bundling and contractile motor proteins. Binding of individual filaments, actin cross-linking and motor proteins allow the formation of thicker, linear and either paralleled or antiparallel filamentous F-actin networks that can be found in all subcellular localisations. In the lamellum, the class II non-muscle myosin family has been shown to be a key regulator, participating in the bundling of actin filaments and generating mechanical forces, which result in filaments sliding and/or contractions [72, 73].
NMMII isoforms in the form of NMMIIA, NNMIIB and NMMIIC are found in most, but not all human cells and most mammals. NMMIIA and NMMIIB are expressed at similar levels in endothelial and epithelial cells. There is, however, little or no NMMIIC present in these cell subsets although it is known to be much more prominent in cells of lung and nervous tissues. Their location appears to be cell-specific and will be regulated differentially depending on the type of cell migration. Perhaps their expression patterns further reflects their function, since NMMIIA and NMMIIB play fundamental roles in mediating cell shape and matrix interactions during migration, whilst a clear role of NMMIIC in this process is still missing. As a consequence, during mesenchymal motility, NMMIIA is localised throughout the cells, in cellular protrusions and in both the lamellipodia and lamella of migrating cells  where, with Arp2/3-dependent actin polymerisation in the former, it results in actomyosin contraction and the retrograde flow of F-actin towards the cell body . NMMIIB is predominantly found in the central and rear regions of the cell but not at the cell front .
Whilst formation of protrusions is a key element of cellular motility, it is equally critical that these newly created extensions also adhere and attach to the substratum, as their inability to do so result in their rearward movements in waves due to cellular tension . Nascent adhesions are the first observable adhesive structures established in the lamellipodium [72, 81]. Their transient nature will force them to either disassemble quickly or mature into larger complexes known as focal adhesions which reside at the boundary of the lamellipodium and lamellum . Actomyosin contraction is the main regulator that controls nascent adhesion enlargement . This initial interaction with the extracellular environment will in turn induces activation of downstream effectors transmitted to the plasma membrane to encourage further integrin adhesion and clustering through their activation as well as changes in confirmation of extracellular matrix proteins . Following on from this integrin activation, other downstream cellular pathways are also instigated; these lead to the recruitment, in a force dependent manner, of numerous framework and adaptor proteins which associate to form an adhesome . Indeed mechanical stretch generated during this process induces changes in structural protein configurations, thereby encouraging binding of certain factors to other partners, as is the case for paxillin, vinculin, talin and actin, three essential component of the adhesome/adhesion complex [85, 86] (Figure 4).
NMMII cross-linking and contractile functions to remodel the actin cytoskeleton are regulated by phosphorylation of (1) regulatory light chains (RLC), (2) heavy chains (HC) and (3) myosin’s ability to assemble into filaments. The RLC polypeptide can be specifically phosphorylated at different regulatory sites. The most prominent site is Ser19, leading, when phosphorylated, to a significant increase in ATPase activity of the head domain in the presence of actin . Phosphorylation sites in the C-terminus of the myosin heavy chain have also been identified, although the true implications of such changes in ATPase activity remain unclear. Regulation of the reversible phosphorylation of specific residues is obtained through the activation of different kinases and phosphatases, depending on the residues considered. Whilst phosphorylation at residue Ser19 will be regulated by MLCK (myosin light chain kinase) and MYPT1 (myosin phosphatase target subunit 1), the most prominent examples, respectively [88, 89], the C-terminal region of the myosin heavy chain is directly targeted by kinases such as TRPM7 (transient receptor potential cation channel subfamily M member 7) and PKC (protein kinase C) [77, 90].
Compelling evidence also demonstrates that phosphorylation and interactions with the C-terminal region of NMMII regulate assembly of the complex into filaments. Such post-translational modifications also result in a change in overall structure. In its fully dephosphorylated form, the compact NMMII complex adopts an asymmetric state, unable to polymerise which is relaxed into an extended conformation following phosphorylation of RLC . Phosphorylation of the C-terminal region of NMMII by kinases such as TRPM7 and PKC has also been shown to interfere with its abilities to form filaments . Finally, it is predominantly through changes at the C-terminus, via the coiled-coil domain that NMMII assembly into filaments is controlled. Truncation experiments have demonstrated that the C-terminal region containing an ACD (assembly competence domain), as well as the C-terminal tail piece, are both important to promote correct assembly of NMMII into parallel and anti-parallel filaments [93, 94]
Another key regulator of motility is the cell’s ability to orchestrate the different contraction and tension forces that are necessary to promote movement. In this section, lamellum components such as those of the NMMII and tropomyosin networks have been briefly reviewed. The overarching purpose here being once again to focalise the reader’s mind on certain complexes, which are known interactors for the S100A4 proteins and could be key players in the process of motility observed when this protein is aberrantly expressed. Other pivotal factors that regulate motility via contractions have not been mentioned here but have been addressed in numerous other reviews and chapters [23, 95–101].
3. S100A4 protein regulation of the actomyosin cytoskeleton
Initially, referred to as mts1, FSP1, metastatin, p9Ka, PEL98, calvasculin, 42A and placental calcium-binding protein, S100A4 is a low-molecular weight acidic protein belonging to the S100 family. This small polypeptide is characterised, as all other members of this family, by a pair of calcium-binding helix-loop-helix regions referred to as EF-hand calcium-binding domains located at either side of the protein and separated by a hinge region [16, 102]. Over the years, S100A4 has received a large degree of interest in the field of cancer cell biology, since its expression is linked to increased motility and invasion directly promoting metastasis in animals, and it is now considered a potent marker for human metastasis and predictor for poor patient outcomes [17, 103]. Its expression is also observed in non-physiopathological states in different motile cell types in vivo, including those of the immune system (lymphocytes, macrophages and neutrophils) as well as mesenchymal fibroblastic cells. The biological implications of S100A4 expression on cellular migration are well-known , but the mechanisms required to attain such phenotypic changes are not fully characterised. In this part of the chapter, I will review its interactions with the different actomyosin components that have been discussed, whether actin nucleating activators, actin binding proteins or myosin regulators, highlighting how their functions are being affected in the presence of the S100A4 protein.
3.1. S100A4 regulates cellular protrusion
Expression of S100A4 has been correlated to significant changes in overall actin organisation and cellular extensions, with extensive increases in lamellipodia and forward protrusions [104, 105] which, in turn, are thought to result in greatly enhanced cellular motility. Similarly, S100A4 has been found to be enriched at both the leading edge and in pseudopodia of migrating cells [104, 106]. A clear molecular explanation as to how S100A4 can regulate such processes is currently lacking, but different hypotheses have been formulated in view of what is known about its different binding partners and regulatory functions.
Recent evidence indicates that the retrograde flow exerted by NMMII impedes extension of the leading edge and subsequent rates of protrusions because of reduced actin polymerisation [130, 131]. Therefore, a reduction in contractile forces, as observed when NMMII activity in cells is inhibited by blebbsistatin, leads to significant decreases in actin retrograde flow in the lamellum. This reduced flow leads, in turn, to subsequent increases in actin clustering into bundles at the lamellipodium–lamellum interface, and increased leading edge extension [132, 133]. The opposite experiment where activation of the NMMII complex is achieved through a Eph receptor/RhoA/ROCK signalling pathway was found to inhibit lamellipodial extension .
It is therefore rational to suggest that one of the protrusion-promoting abilities of S100A4 may be directly linked to its ability to interfere with formation of myosin fibres and their role in protrusion formation. Experimental support for a S100A4-NMMIIA role in cellular protrusion was obtained when S100A4 overexpression in tumour cells led to significant increase in leading edge protrusive activity . This change in phenotype was shown indirectly to be NMMIIA dependent since the exogenous addition of antibody targeted towards the NMMIIA-binding site mimicked the cellular behaviour . Interestingly, a reverse experiment also confirmed a clear role for S100A4 in this process, since S100A4 (−/−) macrophages demonstrated large amounts of over assembled NMMIIA filaments, leading to significant lamellipodia instability and reduced persistence . Because NMMIIA has been clearly demonstrated to be the preferential cytoskeletal target for S100A4 to date, it is expected that their partnership may also influence the overall organisation of the actomyosin network in the lamellipodium and lamellum where the majority of the NMMIIA pools are located.
Whilst S100A4 expression in cells has undoubtedly been linked to regulation of cellular protrusions, mainly increases in lamellipodia and forward protrusions, and arguably being responsible at least in part for some of the enhanced cellular motility observed, the true molecular processes responsible are yet to be fully characterized. In this section, S100A4 partners such as Rhotekin, actin or liprin possibly found at the leading edge are presented in order to provide possible explanations as to how such biological properties may be regulated and it is expected that future researches will shed new lights into the true mechanisms that are involved in this process.
3.2. S100A4 functions in the lamellum and cellular contractions
Behind the cellular protrusion of the highly dynamic lamellipodium, the actomyosin network contributes to cell migration through contractibility and substrate adhesion [61, 72, 81]. Although direct evidence of S100A4 being localised in the lamella of migrating cells is still lacking, numerous reports have highlighted potential regulatory functions of the S100A4 within this subcellular fraction since its expression has been shown to lead to dramatic changes in numbers and organisation of focal adhesions and actin stress fibres [104, 105, 109, 122, 128, 136]. I will briefly discuss here the different properties that S100A4 encompasses towards a remodelling of this cellular architecture.
This final section briefly summarizes the best characterized and known S100A4 interactor, namely NMMII, in view of their high binding affinity, as well as by the number of reports highlighting their association. Yet again, whilst we are gathering further understanding related to their association and the different regions of the proteins that are responsible, a true model as to how their interactions regulate cellular motility remains elusive both theoretically and more importantly experimentally.
Controlling the actin cytoskeleton and the motility process is a key function, that when going awry, leads to significant pathological conditions. Not surprisingly, mutations or aberrant expression of all actin interacting proteins listed in this section have been liked to diseases, thought to be the result of significantly reduced cellular motility. Mutations in cytoplasmic actin have been related to autosomal dominant hearing loss . Mutation or molecular mechanisms that result in changes of activity of actin-binding proteins, such as the nucleating facilitating protein complex Arp2/3 have been linked, albeit indirectly, to bacillary dysentery, as their functions are high jacked by the
I would like to apologise for the numerous studies, which have significantly improved our understanding of actin dependent processes regulating cellular motility and invasion, but could not be included in this work owing to limits on the number of references. Special thanks are given to Prof. Philip S. Rudland (the University of Liverpool) for his critical comments on the chapter and for Prof. David Poyner (Aston University) for his invaluable help with the preparation of the different actin structures used.
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