Mechanosignaling

What is the first step in focal adhesion assembly?

Focal adhesion formation is initiated upon the binding of adhesion receptors to extracellular matrix (ECM) ligands (e.g. fibronectin, vitronectin, collagen) along the cell periphery usually at the protruding edge of a cell. Both intracellular and extracellular factors can influence the level of matrix binding, in terms of affinity (the strength of interactions, reviewed in [1][2]) and avidity (the number of interactions, such as lateral interactions between independently activated proteins within a focal adhesion). Nascent focal adhesions first appear exclusively in the lamellipodium as submicron-sized puncta that are typically immobile but can at times travel short distances along the direction of the actin retrograde flow [3][4].

As the primary ECM receptor in the focal adhesions, integrins are heterodimeric transmembrane proteins, with large multidomain extracellular portions and small cytoplasmic tails. Although there are many different types of integrins with specificity to different ECM, a major portion of cellular and biophysical studies have focused on fibronectin-binding α5β1 and αvβ3 integrins. β1 integrins have been shown to exhibit catch bond behavior[5][6] and function as the force-bearing component. In this capacity β1 integrins maintain adhesion strength despite fluctuating matrix forces, which can often change quite rapidly. However, it is unclear whether β1 integrins bind matrix molecules at the leading edge and translocate inwards [7][8] or get recruited at later stages [9][10]. The less stable β3 integrins are responsible for initiating mechanotransduction and reinforcing focal adhesion attachments to the ECM, in complex with talin [11]. Existence of a phosphorylation-dependent crosstalk between the two integrin types during migration has also been reported [12].

Upon binding of ligands by integrins and clustering, a number of signal transduction cascades are activated. One key event is Rac1 activation and consequent phosphoinositide production [13][14], which leads to the recruitment of the talin homodimer [15][16]. Recent studies provide evidence that anchoring of talin at focal adhesions also requires F-actin and vinculin [17]. This is followed by integrin recruitment and activation upon fibronectin binding [18]. The talin-mediated linkage to the actin cytoskeleton serves to stabilize the integrin-ECM bonds [7]. As talin is pulled by the moving actin, it either stretches leading to rearward translocation of β1 integrins and unfolding of fibronectin [19] or results in frequent slip bonds of 2 pN on immobilized β3 integrins [20][21]. Thus, a dynamic nanoscale organization of integrins exists inside focal adhesions, determined by their extracellular domains [18]. Further, this study also reports a distinct dynamics of integrins within focal adhesions, where they constantly alternate between ligand-bound activated and unbound inactivated states, which is thought to confer the adaptability to focal adhesions in order to withstand rapid changes in force [22].

Talin is one of the best characterized focal adhesion proteins that play important roles during focal adhesion initiation. In addition to its binding site to integrin cytoplasmic tails that can activate integrin (reviewed in [1]), talin can also bind directly to actin and associate with numerous cytoskeletal and signaling proteins (reviewed in [23]), effectively forming one of the core cell-ECM mechanotransduction units.

With the integrin considered ‘engaged’, the adhesion complexes are capable of exerting low level forces to fibronectin, leading to a cascading series of events:

1. The rearrangement of ECM ligand domains to adjust the length of talin
2. The strengthening of the talin-actin slip bond [20]
3. The slowing down of the actin retrograde flow which also helps to prevent the disintegration of the nascent adhesion complexes [3].

In addition to integrins, syndecan-4 also plays an essential role [24][25], binding different domains of matrix proteins and eliciting cooperative signals (reviewed in [26]). Several membrane proteins are also believed to be interacting with integrins, likely as co-receptors, although their functions in cell migration and mechanotransduction remain not well understood (reviewed in [27]).

It should be noted that nascent adhesions do not uniformly undergo these sequential growth. Rather, shear forces generated by actin retrograde flow result in the disassembly of certain fraction of nascent adhesions, and only a subset of nascent adhesions survive to proceed into later stages.

References

1. Calderwood DA. Integrin activation. J. Cell. Sci. 2004; 117(Pt 5):657-66. [PMID: 14754902]
2. Harburger DS, and Calderwood DA. Integrin signalling at a glance. J. Cell. Sci. 2009; 122(Pt 2):159-63. [PMID: 19118207]
3. Alexandrova AY, Arnold K, Schaub S, Vasiliev JM, Meister J, Bershadsky AD, and Verkhovsky AB. Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow. PLoS ONE 2008; 3(9):e3234. [PMID: 18800171]
4. Choi CK, Vicente-Manzanares M, Zareno J, Whitmore LA, Mogilner A, and Horwitz AR. Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 2008; 10(9):1039-50. [PMID: 19160484]
5. Kong F, García AJ, Mould AP, Humphries MJ, and Zhu C. Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 2009; 185(7):1275-84. [PMID: 19564406]
6. Friedland JC, Lee MH, and Boettiger D. Mechanically activated integrin switch controls alpha5beta1 function. Science 2009; 323(5914):642-4. [PMID: 19179533]
7. Nishizaka T, Shi Q, and Sheetz MP. Position-dependent linkages of fibronectin- integrin-cytoskeleton. Proc. Natl. Acad. Sci. U.S.A. 2000; 97(2):692-7. [PMID: 10639141]
8. Puklin-Faucher E, and Sheetz MP. The mechanical integrin cycle. J. Cell. Sci. 2009; 122(Pt 2):179-86. [PMID: 19118210]
9. Laukaitis CM, Webb DJ, Donais K, and Horwitz AF. Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells. J. Cell Biol. 2001; 153(7):1427-40. [PMID: 11425873]
10. Zaidel-Bar R, Cohen M, Addadi L, and Geiger B. Hierarchical assembly of cell-matrix adhesion complexes. Biochem. Soc. Trans. 2004; 32(Pt3):416-20. [PMID: 15157150]
11. Roca-Cusachs P, Gauthier NC, Del Rio A, and Sheetz MP. Clustering of alpha(5)beta(1) integrins determines adhesion strength whereas alpha(v)beta(3) and talin enable mechanotransduction. Proc. Natl. Acad. Sci. U.S.A. 2009; 106(38):16245-50. [PMID: 19805288]
12. Blystone SD, Slater SE, Williams MP, Crow MT, and Brown EJ. A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function. J. Cell Biol. 1999; 145(4):889-97. [PMID: 10330414]
13. Tolias KF, Cantley LC, and Carpenter CL. Rho family GTPases bind to phosphoinositide kinases. J. Biol. Chem. 1995; 270(30):17656-9. [PMID: 7629060]
14. Auer KL, and Jacobson BS. Beta 1 integrins signal lipid second messengers required during cell adhesion. Mol. Biol. Cell 1995; 6(10):1305-13. [PMID: 8573788]
15. Martel V, Racaud-Sultan C, Dupe S, Marie C, Paulhe F, Galmiche A, Block MR, and Albiges-Rizo C. Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides. J. Biol. Chem. 2001; 276(24):21217-27. [PMID: 11279249]
16. Campbell ID, and Ginsberg MH. The talin-tail interaction places integrin activation on FERM ground. Trends Biochem. Sci. 2004; 29(8):429-35. [PMID: 15362227]
17. Banno A, Goult BT, Lee H, Bate N, Critchley DR, and Ginsberg MH. Subcellular localization of talin is regulated by inter-domain interactions. J. Biol. Chem. 2012; 287(17):13799-812. [PMID: 22351767]
18. Rossier O, Octeau V, Sibarita J, Leduc C, Tessier B, Nair D, Gatterdam V, Destaing O, Albigès-Rizo C, Tampé R, Cognet L, Choquet D, Lounis B, and Giannone G. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 2012; 14(10):1057-67. [PMID: 23023225]
19. Smith ML, Gourdon D, Little WC, Kubow KE, Eguiluz RA, Luna-Morris S, and Vogel V. Force-induced unfolding of fibronectin in the extracellular matrix of living cells. PLoS Biol. 2007; 5(10):e268. [PMID: 17914904]
20. Jiang G, Giannone G, Critchley DR, Fukumoto E, and Sheetz MP. Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin. Nature 2003; 424(6946):334-7. [PMID: 12867986]
21. Giannone G, Mège R, and Thoumine O. Multi-level molecular clutches in motile cell processes. Trends Cell Biol. 2009; 19(9):475-86. [PMID: 19716305]
22. Ivaska J. Unanchoring integrins in focal adhesions. Nat. Cell Biol. 2012; 14(10):981-3. [PMID: 23033047]
23. Critchley DR, and Gingras AR. Talin at a glance. J. Cell. Sci. 2008; 121(Pt 9):1345-7. [PMID: 18434644]
24. Longley RL, Woods A, Fleetwood A, Cowling GJ, Gallagher JT, and Couchman JR. Control of morphology, cytoskeleton and migration by syndecan-4. J. Cell. Sci. 1999; 112 ( Pt 20):3421-31. [PMID: 10504291]
25. Zimerman B, Volberg T, and Geiger B. Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading. Cell Motil. Cytoskeleton 2004; 58(3):143-59. [PMID: 15146534]
26. Morgan MR, Humphries MJ, and Bass MD. Synergistic control of cell adhesion by integrins and syndecans. Nat. Rev. Mol. Cell Biol. 2007; 8(12):957-69. [PMID: 17971838]
27. Geiger B, Bershadsky A, Pankov R, and Yamada KM. Transmembrane crosstalk between the extracellular matrix–cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2001; 2(11):793-805. [PMID: 11715046]