Focal Adhesions, Stress Fibers and Mechanical Tension

Experimental Cell Research 343 (2016) 14–20

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Experimental Cell Research

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Review Article Focal adhesions, stress ﬁbers and mechanical tension

Keith Burridge a,n, Christophe Guilluy b a Department of Cell Biology and Physiology, and Lineberger Comprehensive Cancer Center, 12-016 Lineberger, CB#7295, University of North Carolina, Chapel Hill, NC, USA b Inserm UMR_S1087, CNRS UMR_C6291, L'institut du Thorax, and Université de Nantes, Nantes, France article info abstract

Article history: Stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical Received 5 October 2015 tension. Evidence accumulated over many years led to the conclusion that mechanical tension generated Accepted 23 October 2015 within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal Available online 28 October 2015 adhesions. However, several lines of evidence have recently been presented against this model. Here we Keywords: discuss the evidence for and against the role of mechanical tension in driving the assembly of these Myosin II structures. We also consider how their assembly is influenced by the rigidity of the substratum to which RhoA cells are adhering. Finally, we discuss the recently identified connections between stress fibers and the Integrins nucleus, and the roles that these may play, both in cell migration and regulating nuclear function. Substratum rigidity & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction...... 14 2. Systems for analyzing SF and FA assembly...... 15 3. The role of myosin and tension in the development of stress ﬁbers and focal adhesions...... 15 4. Evidence against tension playing a dominant role in FA and SF assembly ...... 17 5. Reconciling the differences ...... 17 6. Stress ﬁbers, focal adhesions and rigidity of the substratum ...... 18 7. Connections to the nucleus...... 18 8. Concluding remarks...... 19 Acknowledgments...... 19 References...... 19

1. Introduction their role in cell migration, and how they assemble and disassemble. Much evidence has indicated that these structures are Growing in culture many cell types, particularly those of me- mechanosensitive [1–5] and it was concluded earlier that me- senchymal origin, display prominent bundles of filamentous actin chanical tension contributes to their assembly [1,6,7]. However, (F-actin) associated with myosin II, α-actinin and several other several recent studies have challenged this view and demonstrated cytoskeletal proteins. These structures, known as stress fibers (SFs) a more complex situation [8–11]. Here, we consider the role of occur in several distinct forms. Frequently, they are associated at mechanical force in the assembly of SFs and FAs. one or both ends with adhesions to the underlying matrix, known It is often forgotten, even by those who study FAs and SFs, that most commonly as focal adhesions (FAs). For over 40 years there these structures are not needed for cell migration [1,12]. Many has been considerable interest in the functions of these structures, cells (e.g. leukocytes) do not develop FAs or SFs but migrate highly effectively. Indeed, the presence of FAs can hinder cell migration due to excessive adhesion. Nevertheless, many migratory cells do n Corresponding author. E-mail addresses: [email protected] (K. Burridge), display FAs and SFs. In these cells there must be a dynamic cou- [email protected] (C. Guilluy). pling of adhesion strength and traction force for cells to move http://dx.doi.org/10.1016/j.yexcr.2015.10.029 0014-4827/& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). K. Burridge, C. Guilluy / Experimental Cell Research 343 (2016) 14–20 15 forward. It is important that both adhesion and traction at the recapitulate the events that occur as cells migrate and engage the front are stronger than at the rear, and so mechanisms must exist ECM at new sites. for modulating these. Force at the front of migrating cells derives Cell migration involves a series of transitions that affect both from both retrograde actin flow and myosin-generated tension [4]. the adhesions and organization of the actin cytoskeleton. As the For many years, the term stress fiber was most commonly ap- lamellipodium extends, driven by Arp2/3 complex-mediated actin plied to the large bundles of F-actin that traverse much of the cell polymerization, initial adhesions form as integrin receptors en- and that are anchored at both ends by FAs. However, it was readily gage the underlying matrix [4,23]. Such “nascent adhesions” are apparent that a variety of structures were often referred to as often transient and many rapidly disassemble. Maturation of ad- stress fibers. In 1998, Small and colleagues distinguished 2 types of hesions that are not disassembled occurs at the transition between SF: ventral SFs, which are anchored at each end by a FA, and dorsal the lamellipodium and lamella, where retrograde actin flow SFs, which are anchored only at one end by a FA close to the cell changes from being driven by actin polymerization to myosin- front. Dorsal SFs extend back toward the nucleus and upwards based contraction. Whereas actin is organized in the lamellipo- toward the dorsal cell surface. They also discussed “arcs”, convex dium as a branching dendritic network, in the lamella it is often bundles of F-actin that form behind the leading edge of migrating bundled into the different SF types [24]. The maturing adhesions or spreading cells and which move rearwards below the dorsal elongate in the direction of retrograde actin flow and retard the surface [13]. Subsequent studies have included arcs, often referred rate of rearward movement of actin [25]. They act as “molecular to as transverse arcs, as a form of SF [14]. Arcs contain many of the clutches” that couple the force of retrograde flow into forward same proteins, but unlike other types of SF, they are not directly extension of the lamellipodium [4]. Consistent with this clutch- anchored by adhesions to the matrix. However, arcs can give rise like function, it was observed that in stationary cells FAs are often to ventral SFs and will be considered here as a form of SF. pulled toward the nucleus, whereas in migrating cells they are One complicating factor in the relationship between mechan- stationary [26]. The small maturing adhesions are often referred to ical tension and the assembly of FAs and SF is that the three types as “focal complexes” [27]. Some continue enlarging to become of filament bundle collectively referred to as SFs differ in their classical “focal adhesions”. However, the different types of adhe- genesis, behavior, and relationship to FAs. Additionally, different sion are poorly defined and it is often difficult to distinguish one models have been used to study how SFs and FAs assemble. type from another. In general, FAs are dependent on RhoA activity, whereas focal complexes are dependent on active Rac1 or Cdc42 [27]. 2. Systems for analyzing SF and FA assembly Hotulainen and Lappalainen used live cell imaging to analyze assembly of the different SF types in migrating osteosarcoma cells Most studies have examined the assembly and disassembly of [14]. They observed dorsal SFs initiating at small adhesions these structures as cells spread and migrate on coverslips coated forming behind the leading edge. As the cell front extended away with extracellular matrix (ECM) (most commonly fibronectin) from the adhesion, dorsal SFs elongated. This SF growth was in- [4,14–17]. This system is well suited to analyzing adhesion dy- hibited by depleting cells of the formin mDia1. Alpha-actinin was namics and actin organization as cells migrate. The second ex- incorporated into the growing SF. Recruitment of myosin II into the perimental model for examining FA and SF assembly was pio- dorsal SF was a relatively late event. A subsequent study using neered by Ridley and Hall [18]. They exploited the observation that higher resolution imaging concluded that little if any myosin II is some cells lose their SFs and FAs when deprived of serum to be- incorporated into dorsal SFs [16]. Transverse arcs arose behind the come quiescent. Upon re-addition of serum or other factors that lamellipodium from the combination of short myosin filaments activate RhoA, FAs and SFs rapidly reassemble. It should be noted plus actin filaments generated at the leading edge by the Arp2/3 that these cells are usually in a non-migratory state and often complex. In this system, ventral SFs developed most commonly confluent. This system was used to identify RhoA as a key reg- from the fusion of each end of an arc with a dorsal SF. The an- ulatory protein controlling the assembly of these structures. It was nealing of two dorsal SFs growing from opposite sides of a cell also also the system used to show that RhoA-induced assembly of FAs gave rise to ventral SFs, again anchored at each end by FAs. A and SFs was blocked by a variety of inhibitors of myosin activity subtype of ventral SFs (discussed later) is the perinuclear “actin and contractility. This led to the conclusion that RhoA-stimulated cap”, where ventral SFs wrap over nuclei and anchor to elongated myosin activity drives the assembly of SFs and FAs [7]. The FAs [17]. The different SF types are illustrated in Fig. 1. bundling of F-actin to form a SF was attributed not only to the Burnette and colleagues used the same cells to explore the tension generated by myosin but also to myosin's crosslinking of factors that maintain the lamella flat as cells migrate [16]. They F-actin [7,19]. Contractility inhibitors available at the time of these determined that dorsal SFs, which they found contained little to experiments were relatively non-specific. However, subsequent no myosin II, acted as struts connecting the ventral adhesions with studies using blebbistatin and Y27632, which inhibit the activities the dorsal contractile actin meshwork. Their analysis revealed that of myosin II and ROCK respectively, have also shown that in- contraction of transverse arcs generated tension on dorsal SFs, and hibiting myosin activity blocks the formation of most FAs and SFs this caused the dorsal SFs to pivot, thereby flattening the lamella [20,21]. Similarly, knockout or knockdown of myosin II expression [16]. prevented maturation of nascent adhesions into FAs [21,22]. In one study this was found for both myosin IIA and IIB [22], whereas in another this was dependent on knockout of myosin IIA but not IIB 3. The role of myosin and tension in the development of stress [21] fibers and focal adhesions This second model system (stimulation of quiescent cells with serum or Rho-activating factors) was well suited to microinjection Besides the evidence that blocking Rho-mediated myosin ac- of constitutively active or dominant negative constructs. In addi- tivity inhibited ventral SF and FA assembly in quiescent cells [7], tion, it has the advantage of allowing synchronous assembly of FAs support for mechanical tension stimulating assembly comes from and SFs to be studied in many cells. However, many cell types are several observations. For example, shear stress at levels equivalent resistant to serum-starvation; they either maintain FAs and SFs, or to that experienced in arteries induced endothelial cells in culture show only a slight decrease in these structures when deprived of to develop SFs [28]. Direct evidence for mechanical tension sti- serum. Another disadvantage is that this system does not mulating growth of FAs came from Riveline and colleagues who 16 K. Burridge, C. Guilluy / Experimental Cell Research 343 (2016) 14–20

requires both tension and actin polymerization, with each being driven by active RhoA. Supporting this idea that growth of FAs reflects the tension applied to them, Geiger's group used traction force microscopy to reveal a close correlation between the size of FAs and the force that is transmitted across them [29,30]. If mechanical force promotes the growth and maturation of FAs, one prediction is that new components will be recruited to FAs in response to force. This is indeed what was seen both when individual components such as vinculin were examined [31],as well as when the FA proteome was analyzed [32,33]. Examining the composition of isolated FAs, many proteins were found to be recruited to FAs in the presence, but not the absence of active myosin [32,33]. The idea that tension stimulates FA growth and Nucleus maturation is also supported by experiments using beads coated with integrin ligands to apply force to cells. Reinforcement of the adhesion made to beads has been observed in multiple studies [31,34–37]. FA proteins are recruited to the bead adhesion sites under tension [31], again consistent with force contributing to adhesion assembly. Leading arcs What is the basis for the stiffening or reinforcement that occurs edge perinuclear actin cap when tension is applied exogenously on integrins? One of the focal adhesions signaling pathways activated in response to tension on integrins is the RhoA pathway [36–38]. Not only will this lead to increased actin polymerization via the RhoA-activated formin mDia1 [39], but it will also promote the stability of actin filaments by inhibiting the severing activity of cofilin (via 4 4 fi Leading ROCK LIMK phosphorylation of co lin). Additionally, the acti- edge vation of myosin II by ROCK [40,41] will lead to a positive feedback loop increasing tension. Just as importantly, activated myosin II Nucleus that has assembled into bipolar filaments is a multivalent cross- linker and bundler of F-actin [8,19]. Additionally, F-actin reveals fi Fig. 1. Types of stress ber in migrating cells. Schematic representation of SFs in enhanced affinity for myosin II when under tension [42]. Similarly, motile cells, (a) top and (b) side views. Four categories of SFs are observed: dorsal fi SFs, transverse arcs, ventral SFs and the perinuclear actin cap. in response to tension, F-actin binds less co lin and is more resistant to severing by cofilin [43]. applied force directly to individual cells with a glass rod. They How might force on a FA lead to recruitment of new components? One way may involve exposure of tension-sensitive cryptic observed growth of adhesions by IRM optics and incorporation of protein binding sites (illustrated in Fig. 2A). Originally demon- fluorescently labeled FA proteins [6]. Adhesion growth was strated for fibronectin [44], this has also been shown for proteins blocked by inhibiting RhoA activity, but this could be rescued by at the cytoplasmic face of FAs, such as talin. Applying tension to expression of the constitutively active formin, mDia1, which is single talin molecules exposed previously buried vinculin binding normally activated by RhoA. It was concluded that FA growth sites [45]. Similarly, other proteins may expose sites that become

myosin-generated contractility Myosin II activity

actin Rock 1 polymerization protein mDia partner RhoA RhoA

Tension 2 p115/LARG GEF-H1/LARG unstretched kinase protein 1 2

resistance from the matrix β1 αv

Extracellular matrix

Fig. 2. Mechanical tension activates signaling pathways at adhesion sites. a. Exposure of cryptic sites. Tension can affect protein conformation to expose protein binding sites (e.g. talin) or sites for kinases or other protein modifications (e.g. p130cas). b. RhoA regulation of adhesion maturation through Rock and mDia. Adhesion to fibronectin activates RhoA through p115 RhoGEF and LARG [91], leading to Rock-mediated myosin stimulation. In response to tension, GEF-H1, LARG and mDia are recruited [32,37] and promote actin polymerization. Β1 (1) and αv (2) integrin subtypes cooperate to regulate RhoA signaling and adhesion maturation [62]. K. Burridge, C. Guilluy / Experimental Cell Research 343 (2016) 14–20 17 modified, thereby facilitating new interactions [46]. For example, in these studies is that these last two groups examined FAs in mechanically stretching p130cas exposed multiple tyrosines that migrating cells, whereas the study reporting a positive correlation could be phosphorylated by Src [47]. between FA size and tension used stationary cells [29]. Another SFs have also been shown to undergo reinforcement in re- study found that for larger FAs their size was proportional to the sponse to strain. First demonstrated when cells experience cyclic force being transmitted, but for small adhesions the correlation stretch or shear stress [48], zyxin shuttles from FAs to zones of broke down [56]. Since small adhesions are usually at the cell tension within SFs and triggers local recruitment of α-actinin and front, a potential explanation is that the additional force experi- vasodilator-stimulated phosphoprotein (VASP) that thickens and enced by these small adhesions arises from retrograde actin flow reinforces SFs [48,49]. Zyxin-dependent SF reinforcement has also and from transverse arcs that transmit tension to the matrix via been observed in cells that are not subjected to external me- dorsal SFs. However, this does not explain why these small adhe- chanical stress. This repair mechanism serves to limit SF elonga- sions do not enlarge in response to the high force being applied, tion and breakage at sites of excessive strain [50]. Interestingly, which would be predicted by the Riveline study [6]. zyxin targeting to SFs depends on MAPK activation. Although the Choi and colleagues investigated the role of myosin II in nas- mechanism involved is not fully understood, multiple models have cent adhesion maturation [8]. Initial adhesion assembly occurred been proposed and hinge on tension-dependent conformational within the lamellipodium independently of myosin II on a tem- changes in SF-associated proteins to expose new docking sites for plate, the dorsal SF. The rate of assembly was proportional to the zyxin [50]. rate of protrusion of the lamellipodium. Template formation re- Little is known about how the bonds between particular pro- quired α-actinin, indicating the importance of F-actin crosslinking. teins in FAs respond to mechanical tension. However, in some The subsequent maturation of these nascent adhesions into FAs cases “catch” bonds, which strengthen in response to tension [51], was blocked by knockdown of myosin IIA expression. Strikingly, have been identified, for example, for the fibronectin-binding in- the cells depleted of myosin IIA could be rescued by inactive motor tegrin α5β1 [52,53]. We suspect that more catch bonds will be mutants of myosin IIA that were unable to generate force. How- identified operating in FAs. We anticipate that some of the sig- ever, these mutants maintained their actin-binding ability. It was naling associated with mechanical tension will modify protein concluded that myosin's crosslinking function is more important interactions, converting slip bonds into catch bonds, thereby fa- than its tension-generating function for maturation of nascent cilitating tension-induced stabilization. Conversely, the conversion adhesions [8]. of catch bonds into slip bonds may contribute to the disassembly Some of the strongest arguments against tension driving as- of adhesions at the rear of migrating cells. sembly of FAs have come from Gardel and her colleagues. They demonstrated that in migrating cells up to 60% of the force exerted on the substratum is generated by the lamellar actin network ra- 4. Evidence against tension playing a dominant role in FA and ther than by SFs [59]. Using blebbistatin wash-out as a trigger for SF assembly inducing myosin activity, they showed that the assembly of SFs lagged temporally behind the development of traction forces. They In contrast to the above studies, several have challenged the also noted that FA growth rate remained constant under a range of importance of myosin-generated tension in FA and SF assembly. different tensions [60]. Titrating the level of myosin activity by Some of the different conclusions may reflect that different types varying the concentration of inhibitors led them to conclude that of SF and FAs assemble through different mechanisms. So, for tension is necessary but not sufficient for FA maturation [10]. They example, in the study by Hotulainen and Lappalainen, inhibiting also concluded that the dorsal SF was critical as a structural myosin activity rapidly blocked formation of transverse arcs, but template [10]. had little initial effect on dorsal SFs and the small adhesions that Waterman's group found that FA growth was fast in cells anchor them [14]. Tension was also important in the conversion of lacking vinculin but that these cells exerted low traction on the transverse arcs into ventral SFs, following their fusion with dorsal substratum [11]. They concluded that vinculin when present cou- SFs. The role of myosin in dorsal SF assembly seems minimal. In ples retrograde actin flow to integrins and acts as a molecular several ways, dorsal SFs resemble filopodia. Both structures consist clutch. Their finding that vinculin promotes the transmission of of narrow bundles of F-actin anchored either in a tip complex force to the substratum but results in slower growth of FAs runs (filopodium) or nascent adhesion (dorsal SF). The filaments within counter to the idea that force itself contributes to FA growth. these bundles all appear initially to have a single polarity, with polymerization occurring at the membrane attachment site, i.e. the filopodial tip or matrix adhesion. Because myosin II cannot 5. Reconciling the differences generate contractile force on bundles of F-actin with a single polarity, whenever myosin II incorporates into these structures (such How can these seemingly different conclusions be reconciled? as when dorsal SFs are fusing with transverse arcs), then the po- Some of the differences may come from the use of the different larity of the actin filaments must be broken so that the bundle systems, migrating cells versus quiescent stationary cells. Other contains F-actin of opposite polarities. Little is known about how differences surely derive from grouping together the different this polarity transition occurs, although some possibilities have types of SF and adhesion that are seen in migrating cells. The or- been suggested [5,14]. ganization and behavior of dorsal SFs is different from transverse Tension generated within stress fibers is transmitted across FAs arcs and ventral SFs. The former seem largely independent of as demonstrated by studying traction forces at a subcellular scale myosin-generated contractility whereas myosin is important for [29,54–57] and shown most directly using a vinculin tension both transverse arcs and ventral SFs. However, myosin is needed sensor [58]. As mentioned above, Geiger and his colleagues cor- for the maturation of adhesions, particularly its crosslinking related the magnitude of force generated at a FA with the cross- function as shown by Choi and colleagues [8]. sectional area of the FA [29]. A very different conclusion was In the original model of tension driving SF and FA assembly, reached by Beningo and colleagues who found that small adhe- actin polymerization was relatively minor [7]. The pulling together sions at the front of a migrating cell developed greater tractional and alignment of pre-existing filaments, already attached to in- force than the larger adhesions further away from the front [55]. tegrins, was more important than nucleation of new filaments. Similar results were reported by Stricker et al. [9]. A key difference This was supported by limited actin polymerization as SFs and FAs 18 K. Burridge, C. Guilluy / Experimental Cell Research 343 (2016) 14–20 assembled in the quiescent cell model [61]. However, it is clear the substratum. that actin polymerization is essential for the development of SFs and FAs in migrating cells [10,14]. One possibility is that mechanical tension may stimulate formin-mediated actin poly- 7. Connections to the nucleus merization at FAs. Such a mechanism was suggested in the Rive- line study [6], in which they observed that tension-dependent It has long been known that tension exerted on the cell surface adhesion growth was dependent on mDia. Consistent with this, is transmitted to the nucleus and that multiple connections link Schiller and colleagues recently observed that actomyosin con- the nucleus to the cytoskeleton [70]. Indeed, mechanical force tractility was necessary to recruit mDia to adhesions [62]. Using a exerted on cells has profound effects on gene transcription [71– proteomic approach, they discovered that β1 and αv integrins 74], and tension exerted via SFs impacts nuclear structure and cooperate to activate RhoA during adhesion to fibronectin but that function [75,76]. The nesprin family of proteins span the outer downstream of RhoA the pathways diverged. Unexpectedly, β1 nuclear membrane, connecting the different filament systems to integrins were coupled to ROCK and activation of myosin II, proteins spanning the inner nuclear membrane such as SUN 1 and whereas αv was coupled to activation of mDia and actin poly- 2 [74,77,78]. These proteins connecting the nucleus with the cy- merization. However, in response to myosin II activity and high tension, αv integrin was recruited along with mDia to the adhe- toskeleton have been referred to as the Linker of nucleoskeleton sions. They concluded that cooperation between these fibronectin- and cytoskeleton (LINC) complex [79]. binding integrins and their RhoA signaling pathways (Fig. 2B) Investigating the relationship of the centrosome and nucleus to fi contributed to adhesion maturation [62]. signals driving directional broblast migration, Gundersen's group observed that the nucleus was pulled rearwards by actin filaments, such that the centrosome came lie in front of it [80]. Pursuing this fi fi 6. Stress fibers, focal adhesions and rigidity of the substratum further, they identi ed bundles of actin laments (dorsal actin cables) moving rearward over the nucleus and forming aligned It has long been recognized that SFs are more prominent in arrays with the LINC complex. These transmembrane actin-asso- cells growing on plastic or glass surfaces than they are in the same ciated nuclear lines (TAN lines) appear closely related to arcs that cells in their native tissue environment or growing in 3D systems arise near the cell front and move backwards via retrograde flow [1]. Is the prominence of SFs and FAs in cells growing in vitro due [81]. However, once these arcs pass over the nucleus, they engage to the two dimensional nature of the substratum, to its rigidity, or and align the LINC complex. The rearward movement of nuclei and both? Fibroblasts cultured in three dimensional collagen gels TAN lines is closely correlated. Disrupting the LINC complex dis- rarely develop SFs. Fibroblasts will actively contract free-floating rupts TAN lines and prevents nuclear movement in response to gels, but if the gels are anchored to prevent contraction to a migration signals [81]. Depletion of emerin, an inner nuclear smaller volume, the cells develop isometric tension and develop membrane protein involved in the interaction of the LINC complex SFs [63–65]. Release of the gels from their attachments to the with the nuclear lamins, caused the TAN lines to slip over im- culture dish results in rapid contraction of the gels and dis- mobile nuclei [82]. assembly of the SFs [63–65]. Examining the relationship between cell and nuclear shape, The role of substrate rigidity in SF and FA assembly was ex- Wirtz's group identified a subset of ventral SFs that wrap over the amined directly by Pelham and Wang, who discovered that as- nucleus. These SFs connect to the nucleus via the LINC complex sembly of these structures was inhibited on soft substrata [66]. and form a perinuclear “actin cap”. These SFs were implicated in How might this occur? With the recognition that active RhoA determining nuclear shape, which was observed to be elongated in drives formation of these structures [18], this raised the question the direction of cell migration in moving cells, but round in sta- whether adhesion to a rigid substratum stimulates RhoA activity. tionary cells [17]. Like other ventral SFs, the SFs of the actin cap are Measuring RhoA activity revealed that RhoA is activated in cells anchored by FAs at each end. However, these FAs and their asso- grown on rigid substrata [67,68]. Experimentally applying tension ciated SFs are distinctive in several respects. The FAs have a larger to engaged integrins was also found to activate RhoA [37,38]. area and are more elongated than the FAs associated with other Dissecting the pathway, Guilluy and colleagues identified both ventral SFs [83]. Together with the SFs of the actin cap, they are GEF-H1 and LARG as guanine nucleotide exchange factors (GEFs) more susceptible to disassembly by the actin depolymerizing drug responding to tension applied to fibronectin-coated beads [37]. Latrunculin B. They are also more dynamic, with several FA com- GEF-H1 was activated downstream from FAK, Ras and the MEK/ ponents having shorter half-lives. Strikingly, the FAs associated ERK pathway, whereas LARG was activated via the tyrosine kinase with the actin cap were the first to disassemble after adhesion to Fyn. Keely's group also found GEF-H1 was activated in cells ad- soft substrata, leading to the conclusion that these FAs are more hering to rigid substrata, but in their case this was attributed to mechanosensitive than most FAs [83]. One has to wonder whether the release of GEF-H1 from microtubules [69]. Using proteomic approaches two groups have observed both GEF-H1 and mDia these actin cap-associated FAs are the same as those described by fl “ ” being recruited to adhesions in response to actomyosin con- Waterman's group, i.e. uctuating in tension to sense the stiff- tractility, suggesting that mechanical tension activates a GEF-H1/ ness of the substratum [54]. Persistent migration was associated RhoA/mDia pathway that may promote adhesion growth [32,62]. with an actin cap, whereas this disassembled when cells paused to High resolution traction force microscopy revealed that traction change direction. Experimentally disrupting the actin cap in- generated at one FA was often independent of the traction being hibited persistent cell migration [84]. generated by an adjacent FA within the same cell [54]. The force The above results raise the question of what the role of the transmitted by an adhesion was either stable over time or it nucleus is in cell migration, a topic beyond the scope of this brief fluctuated. In situations where it fluctuated, this “tugging” at FAs review. However, numerous studies have observed that disrupting was dispensable for their maturation, as well as for directional the LINC complex and hence the connections between the nucleus migration in response to chemotactic or haptotactic cues. How- and the cytoskeleton have major effects on cell migration [78,85]. ever, it was needed for durotaxis, the migration up a stiffness Whereas the nucleus appears important in cell migration, it gradient [54]. The authors concluded that fluctuating tension at should also be remembered that cells lacking a nucleus are capable FAs provides a mechanism by which a cell senses the stiffness of of migrating [86,87]. K. Burridge, C. Guilluy / Experimental Cell Research 343 (2016) 14–20 19

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