Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions

A Corrigendum to this article was published on 06 November 2012

Abstract

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β3-integrins are enriched and stationary within FAs, whereas immobilized β1-integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs.

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Figure 1: β3-integrins are immobilized more frequently and undergo slower free-diffusion inside versus outside FAs.
Figure 2: Integrin immobilization correlates with integrin activation and require both fibronectin and ABPs binding.
Figure 3: Integrins undergo repeated cycles of slow free-diffusion and immobilization within FAs.
Figure 4: Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, spatially restricting integrin immobilization to FAs.
Figure 5: Differential nanoscale organization of β3- and β1-integrins in FAs.
Figure 6: β3- and β1-integrin extracellular domains determine their distinct dynamic nanoscale organizations inside FAs.
Figure 7: Differential transmission of F-actin motion to β3-integrin, β1-integrin and talin.

References

  1. 1

    Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Giannone, G., Mege, R. M. & Thoumine, O. Multi-level molecular clutches in motile cell processes. Trends Cell Biol. 19, 475–486 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Parsons, J. T., Horwitz, A. R. & Schwartz, M. A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 11, 633–643 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Zaidel-Bar, R., Itzkovitz, S., Ma’ayan, A., Iyengar, R. & Geiger, B. Functional atlas of the integrin adhesome. Nat. Cell Biol. 9, 858–867 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Sheetz, M. P., Felsenfeld, D., Galbraith, C. G. & Choquet, D. Cell migration as a five-step cycle. Biochem. Soc. Symp. 65, 233–243 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Giannone, G. et al. Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561–575 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Tkachenko, E. et al. Protein kinase A governs a RhoA-RhoGDI protrusion-retraction pacemaker in migrating cells. Nat. Cell Biol. 13, 660–667 (2011).

    Article  Google Scholar 

  8. 8

    Pollard, T. D. & Borisy, G. G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Kanchanawong, P. et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Zamir, E. et al. Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2, 191–196 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Danen, E. H., Sonneveld, P., Brakebusch, C., Fassler, R. & Sonnenberg, A. The fibronectin-binding integrins α5β1 and αvβ3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis. J. Cell Biol. 159, 1071–1086 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Roca-Cusachs, P., Gauthier, N. C., Del Rio, A. & Sheetz, M. P. Clustering of α(5)β(1) integrins determines adhesion strength whereas α(v)β(3) and talin enable mechanotransduction. Proc. Natl Acad. Sci. USA 106, 16245–16250 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Choquet, D., Felsenfeld, D. P. & Sheetz, M.P. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell 88, 39–48 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Jiang, G., Giannone, G., Critchley, D. R., Fukumoto, E. & Sheetz, M. P. Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin. Nature 424, 334–337 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Giannone, G., Jiang, G., Sutton, D. H., Critchley, D. R. & Sheetz, M. P. Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation. J. Cell Biol. 163, 409–419 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Tadokoro, S. et al. Talin binding to integrin β tails: a final common step in integrin activation. Science 302, 103–106 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Moser, M., Legate, K. R., Zent, R. & Fassler, R. The tail of integrins, talin, and kindlins. Science 324, 895–899 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Shattil, S. J., Kim, C. & Ginsberg, M. H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Meth. 5, 155 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Luo, B. H., Springer, T. A. & Takagi, J. Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand. Proc. Natl Acad. Sci. USA 100, 2403–2408 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Cluzel, C. et al. The mechanisms and dynamics of (α)v(β)3 integrin clustering in living cells. J. Cell Biol. 171, 383–392 (2005).

    CAS  Article  Google Scholar 

  22. 22

    O’Toole, T. E., Ylanne, J. & Culley, B. M. Regulation of integrin affinity states through an NPXY motif in the β subunit cytoplasmic domain. J. Biol. Chem. 270, 8553–8558 (1995).

    Article  Google Scholar 

  23. 23

    Loftus, J. C. et al. A β3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 249, 915–918 (1990).

    CAS  Article  Google Scholar 

  24. 24

    McCleverty, C. J., Lin, D. C. & Liddington, R. C. Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions. Prot. Sci. 16, 1223–1229 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Legate, K. R. & Fassler, R. Mechanisms that regulate adaptor binding to β-integrin cytoplasmic tails. J. Cell Sci. 122, 187–198 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043–1057 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Shroff, H., Galbraith, C. G., Galbraith, J. A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nature Meth. 5, 417–423 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Lata, S., Gavutis, M., Tampe, R. & Piehler, J. Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation. J. Am. Chem. Soc. 128, 2365–2372 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    CAS  Article  Google Scholar 

  30. 30

    Brown, C. M. et al. Probing the integrin-actin linkage using high-resolution protein velocity mapping. J. Cell Sci. 119, 5204–5214 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Hu, K., Ji, L., Applegate, K. T., Danuser, G. & Waterman-Storer, C. M. Differential transmission of actin motion within focal adhesions. Science 315, 111–115 (2007).

    CAS  Article  Google Scholar 

  32. 32

    Kiema, T. et al. The molecular basis of filamin binding to integrins and competition with talin. Mol. Cell 21, 337–347 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Wegener, K. L. et al. Structural basis of integrin activation by talin. Cell 128, 171–182 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Ye, F. et al. Recreation of the terminal events in physiological integrin activation. J. Cell Biol. 188, 157–173 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Zhang, X. et al. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat. Cell Biol. 10, 1062–1068 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Goksoy, E. et al. Structural basis for the autoinhibition of talin in regulating integrin activation. Mol. Cell 31, 124–133 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Banno, A. et al. Subcellular localization of talin is regulated by inter-domain interactions. J. Biol. Chem. 287, 13799–13812 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Martel, V. et al. Conformation, localization, and integrin binding of talin dependon its interaction with phosphoinositides. J. Biol. Chem. 276, 21217–21227 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Saltel, F. et al. New PI(4,5)P2- and membrane proximal integrin-bindingmotifs in the talin head control β3-integrin clustering. J. Cell Biol. 187, 715–731 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Pankov, R. et al. Integrin dynamics and matrix assembly: tensin-dependent translocation of α(5)β(1) integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 148, 1075–1090 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell Sci. 119, 3901–3903 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Huveneers, S., Truong, H., Fassler, R., Sonnenberg, A. & Danen, E. H. Binding of soluble fibronectin to integrin α5β1—link to focal adhesion redistribution and contractile shape. J. Cell Sci. 121, 2452–2462 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Anthis, N. J., Wegener, K. L., Critchley, D. R. & Campbell, I. D. Structural diversity in integrin/talin interactions. Structure 18, 1654–1666 (2010).

    CAS  Article  Google Scholar 

  44. 44

    Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically activated integrin switch controls α5β1 function. Science 323, 642–644 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).

    CAS  Article  Google Scholar 

  46. 46

    Arias-Salgado, E. G. et al. Src kinase activation by direct interaction with the integrin β cytoplasmic domain. Proc. Natl Acad. Sci. USA 100, 13298–13302 (2003).

    CAS  Article  Google Scholar 

  47. 47

    Millon-Fremillon, A. et al. Cell adaptive response to extracellular matrix density is controlled by ICAP-1-dependent β1-integrin affinity. J. Cell Biol. 180, 427–441 (2008).

    CAS  Article  Google Scholar 

  48. 48

    Worth, D. C. et al. αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration. J. Cell Biol. 189, 369–383 (2010).

    CAS  Article  Google Scholar 

  49. 49

    Rantala, J. K. et al. SHARPIN is an endogenous inhibitor of [β]1-integrin activation. Nat. Cell Biol. 13, 1315–1324 (2011).

    CAS  Article  Google Scholar 

  50. 50

    Tanentzapf, G. & Brown, N. H. An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton. Nat. Cell Biol. 8, 601–606 (2006).

    CAS  Article  Google Scholar 

  51. 51

    Himmel, M. et al. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions. J. Biol. Chem. 284, 13832–13842 (2009).

    CAS  Article  Google Scholar 

  52. 52

    Wang, P., Ballestrem, C. & Streuli, C. H. The C terminus of talin links integrins to cell cycle progression. J. Cell Biol. 195, 499–513 (2011).

    CAS  Article  Google Scholar 

  53. 53

    Galbraith, C. G., Yamada, K. M. & Galbraith, J. A. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 315, 992–995 (2007).

    CAS  Article  Google Scholar 

  54. 54

    Margadant, F. et al. Mechanotransduction in vivo by repeated talin stretch-relaxation events depends upon vinculin. PLoS Biol. 9, e1001223 (2011).

    CAS  Article  Google Scholar 

  55. 55

    Del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding. Science 323, 638–641 (2009).

    CAS  Article  Google Scholar 

  56. 56

    Kong, F., Garcia, A. J., Mould, A. P., Humphries, M. J. & Zhu, C. Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 1275–1284 (2009).

    CAS  Article  Google Scholar 

  57. 57

    Smith, M. L. et al. Force-induced unfolding of fibronectin in the extracellular matrix of living cells. PLoS Biol. 5, e268 (2007).

    Article  Google Scholar 

  58. 58

    Rossier, O. M. et al. Force generated by actomyosin contraction builds bridges between adhesive contacts. EMBO J. 29, 1055–1068 (2010).

    CAS  Article  Google Scholar 

  59. 59

    Parsons, M., Messent, A. J., Humphries, J. D., Deakin, N. O. & Humphries, M. J. Quantification of integrin receptor agonism by fluorescence lifetime imaging. J. Cell Sci. 121, 265–271 (2008).

    CAS  Article  Google Scholar 

  60. 60

    Plançon, S., Morel-Kopp, M. C., Schaffner-Reckinger, E., Chen, P. & Kieffer, N. Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIb(β3): effect of β3GFP on αIIb(β3) ligand binding. Biochem. J. 357, 529–536 (2001).

    Article  Google Scholar 

  61. 61

    Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific labelling of cell surface proteins with biophysical probes using biotin ligase. Nature Meth. 2, 99–104 (2005).

    CAS  Article  Google Scholar 

  62. 62

    Rottner, K., Behrendt, B., Small, J. V. & Wehland, J. VASP dynamics during lamellipodia protrusion. Nat. Cell Biol. 1, 321–322 (1999).

    CAS  Article  Google Scholar 

  63. 63

    Racine, V. et al. Multiple-target tracking of 3D fluorescent objects based on simulated annealing. IEEE Int. Symp. Biomed. Imag. 1020–1023 (2006).

  64. 64

    Racine, V. et al. Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells. J. Microsci. 225, 214–228 (2007).

    Article  Google Scholar 

  65. 65

    Izeddin, I. et al. Wavelet analysis for single molecule localization microscopy. Opt. Exp. 20, 2081–2095 (2012).

    CAS  Article  Google Scholar 

  66. 66

    Cheezum, M. K., Walker, W. F. & Guilford, W. H. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81, 2378–2388 (2001).

    CAS  Article  Google Scholar 

  67. 67

    Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. Embo J. 22, 4656–4665 (2003).

    CAS  Article  Google Scholar 

  68. 68

    Annibale, P., Scarselli, M., Kodiyan, A. & Radenovic, A. Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long-lived dark state in the red photoconverted form. J. Phys. Chem. Lett. 1, 1506–1510 (2010).

    CAS  Article  Google Scholar 

  69. 69

    Grunwald, C. et al. Quantum-yield-optimized fluorophores for site-specific labelling and super-resolution imaging. J. Am. Chem. Soc. 133, 8090–8093 (2011).

    CAS  Article  Google Scholar 

  70. 70

    Groc, L. et al. Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. J. Neurosci. 27, 12433–12437 (2007).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank C. Breillat, A. Frouin, D. Bouchet and P. Gonzales for technical assistance; M. P. Sheetz, O. Thoumine, J. Petersen, B. Fourcade, M. Block, L. Duchesne and D.G. Fernig for helpful discussions; M. Humphries, N. Kieffer, J. Wehland, A. Gautreau and P. Kanchanawong for the gift of reagents; P. Legros and C. Poujol (Bordeaux Imaging Center) for STED imaging. We acknowledge financial support from the French Ministry of Research and CNRS, ANR grant Nanomotility (G.G., B.L., O.R.), Fondation ARC pour la Recherche sur le Cancer (O.R.), Conseil Régional Aquitaine, Fondation pour la Recherche Médicale, the ERC Program numbers 232942 Nano-Dyn-Syn (D.C., B.L.) and 235552 Glutraf (D.N.), the Human Frontiers Science Programme (B.L.) and The Ligue National contre le Cancer—équipe labellisée 2010 (C.A-R., O.D.). The research was conducted in the scope of the International Associated Laboratory LIA CAFS.

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O.R. and G.G. conceptualized and performed the sptPALM and STED experiments. V.O., C.L., L.C. and B.L. conceptualized and developed the single-protein-tracking set-up used for ATTO647N. G.G. performed single-protein-tracking experiments using ATTO647N. B.T. designed and generated new protein constructs. V.G. and R.T. designed and synthesized the TrisNTA–ATTO647N. D.N. and J-B.S. developed the sptPALM set-up. J-B.S. developed the analytical tools for sptPALM. V.O., C.L. and L.C. developed the analytical tools for single-protein tracking using ATTO647N. O.D. and C.A-R. contributed valuable scientific advice and developed the β1-integrin–mEOS2 and chimaeric integrin constructs. O.D. performed FACS experiments. L.C., D.C., B.L. and G.G. coordinated the study. O.R. and G.G. wrote the manuscript and Supplementary Information. All authors discussed the results and commented on the manuscript.

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Correspondence to Grégory Giannone.

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Rossier, O., Octeau, V., Sibarita, JB. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat Cell Biol 14, 1057–1067 (2012). https://doi.org/10.1038/ncb2588

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