Skip to main content
SpringerLink
Log in

Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration

  • Protocol
  • First Online:
The Integrin Interactome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2217))

  • 1224 Accesses

Abstract

Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein–protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

Cas9:

CRISPR associated protein 9

CRISPR:

Clustered regularly interspaced short palindromic repeats

DONALD:

Direct optical nanoscopy with axially localized detection

dSTORM:

Direct stochastic optical reconstruction microscopy

FBS:

Fetal bovine serum

FRAP:

Fluorescence recovery after photobleaching

FWHM:

Full width at half maximum

mEos2:

Monomeric Eos2

MSD:

Mean square displacement

PALM:

Photoactivation localization microscopy

SAFe:

Supercritical angle fluorescence emission

SMLM:

Single-molecule localization microscopy

SPT:

Single-particle tracking

TIRF:

Total internal reflection fluorescence

WRC:

Wave regulatory complex

References

  1. Montell DJ, Rorth P, Spradling AC (1992) Slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell 71(1):51–62. https://doi.org/10.1016/0092-8674(92)90265-e

    Article  CAS  PubMed  Google Scholar 

  2. Grinnell F (1992) Wound repair, keratinocyte activation and integrin modulation. J Cell Sci 101(Pt 1):1–5

    CAS  PubMed  Google Scholar 

  3. Abreu-Blanco MT, Verboon JM, Liu R, Watts JJ, Parkhurst SM (2012) Drosophila embryos close epithelial wounds using a combination of cellular protrusions and an actomyosin purse string. J Cell Sci 125(Pt 24):5984–5997. https://doi.org/10.1242/jcs.109066

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Friedl P, Locker J, Sahai E, Segall JE (2012) Classifying collective cancer cell invasion. Nat Cell Biol 14(8):777–783. https://doi.org/10.1038/ncb2548

    Article  CAS  PubMed  Google Scholar 

  5. Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112(4):453–465. https://doi.org/10.1016/s0092-8674(03)00120-x

    Article  CAS  PubMed  Google Scholar 

  6. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302(5651):1704–1709. https://doi.org/10.1126/science.1092053

    Article  CAS  PubMed  Google Scholar 

  7. Ridley AJ (2011) Life at the leading edge. Cell 145(7):1012–1022. https://doi.org/10.1016/j.cell.2011.06.010

    Article  CAS  PubMed  Google Scholar 

  8. Svitkina TM, Borisy GG (1999) Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol 145(5):1009–1026. https://doi.org/10.1083/jcb.145.5.1009

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Chen Z, Borek D, Padrick SB, Gomez TS, Metlagel Z, Ismail AM, Umetani J, Billadeau DD, Otwinowski Z, Rosen MK (2010) Structure and control of the actin regulatory WAVE complex. Nature 468(7323):533–538. https://doi.org/10.1038/nature09623

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70(3):401–410. https://doi.org/10.1016/0092-8674(92)90164-8

    Article  CAS  PubMed  Google Scholar 

  11. Machacek M, Hodgson L, Welch C, Elliott H, Pertz O, Nalbant P, Abell A, Johnson GL, Hahn KM, Danuser G (2009) Coordination of Rho GTPase activities during cell protrusion. Nature 461(7260):99–103. https://doi.org/10.1038/nature08242

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, Danuser G (2004) Two distinct actin networks drive the protrusion of migrating cells. Science 305(5691):1782–1786. https://doi.org/10.1126/science.1100533

    Article  CAS  PubMed  Google Scholar 

  13. Case LB, Baird MA, Shtengel G, Campbell SL, Hess HF, Davidson MW, Waterman CM (2015) Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nat Cell Biol 17(7):880–892. https://doi.org/10.1038/ncb3180

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Giannone G, Dubin-Thaler BJ, Dobereiner HG, Kieffer N, Bresnick AR, Sheetz MP (2004) Periodic lamellipodial contractions correlate with rearward actin waves. Cell 116(3):431–443. https://doi.org/10.1016/s0092-8674(04)00058-3

    Article  CAS  PubMed  Google Scholar 

  15. Giannone G, Dubin-Thaler BJ, Rossier O, Cai Y, Chaga O, Jiang G, Beaver W, Dobereiner HG, Freund Y, Borisy G, Sheetz MP (2007) Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128(3):561–575. https://doi.org/10.1016/j.cell.2006.12.039

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Hotulainen P, Lappalainen P (2006) Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J Cell Biol 173(3):383–394. https://doi.org/10.1083/jcb.200511093

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Koestler SA, Auinger S, Vinzenz M, Rottner K, Small JV (2008) Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front. Nat Cell Biol 10(3):306–313. https://doi.org/10.1038/ncb1692

    Article  CAS  PubMed  Google Scholar 

  18. Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR (2009) Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10(11):778–790. https://doi.org/10.1038/nrm2786

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110(6):673–687. https://doi.org/10.1016/s0092-8674(02)00971-6

    Article  CAS  PubMed  Google Scholar 

  20. Geiger B, Spatz JP, Bershadsky AD (2009) Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol 10(1):21–33. https://doi.org/10.1038/nrm2593

    Article  CAS  PubMed  Google Scholar 

  21. Humphrey JD, Dufresne ER, Schwartz MA (2014) Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol 15(12):802–812. https://doi.org/10.1038/nrm3896

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Legate KR, Fassler R (2009) Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. J Cell Sci 122(Pt 2):187–198. https://doi.org/10.1242/jcs.041624

    Article  CAS  PubMed  Google Scholar 

  23. Orre T, Rossier O, Giannone G (2019) The inner life of integrin adhesion sites: from single molecules to functional macromolecular complexes. Exp Cell Res 379(2):235–244. https://doi.org/10.1016/j.yexcr.2019.03.036

    Article  CAS  PubMed  Google Scholar 

  24. Sun Z, Guo SS, Fassler R (2016) Integrin-mediated mechanotransduction. J Cell Biol 215(4):445–456. https://doi.org/10.1083/jcb.201609037

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Rossier O, Octeau V, Sibarita JB, Leduc C, Tessier B, Nair D, Gatterdam V, Destaing O, Albiges-Rizo C, Tampe R, Cognet L, Choquet D, Lounis B, Giannone G (2012) Integrins beta1 and beta3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat Cell Biol 14(10):1057–1067. https://doi.org/10.1038/ncb2588

    Article  CAS  PubMed  Google Scholar 

  26. Schiller HB, Hermann MR, Polleux J, Vignaud T, Zanivan S, Friedel CC, Sun Z, Raducanu A, Gottschalk KE, Thery M, Mann M, Fassler R (2013) β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments. Nat Cell Biol 15(6):625–636. https://doi.org/10.1038/ncb2747

    Article  CAS  PubMed  Google Scholar 

  27. Moser M, Legate KR, Zent R, Fassler R (2009) The tail of integrins, talin, and kindlins. Science 324(5929):895–899. https://doi.org/10.1126/science.1163865

    Article  CAS  PubMed  Google Scholar 

  28. Shattil SJ, Kim C, Ginsberg MH (2010) The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 11(4):288–300. https://doi.org/10.1038/nrm2871

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Bouvard D, Pouwels J, De Franceschi N, Ivaska J (2013) Integrin inactivators: balancing cellular functions in vitro and in vivo. Nat Rev Mol Cell Biol 14(7):430–442. https://doi.org/10.1038/nrm3599

    Article  CAS  PubMed  Google Scholar 

  30. Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, Humphries MJ (2009) Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci Signal 2(87):ra51. https://doi.org/10.1126/scisignal.2000396

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Schiller HB, Friedel CC, Boulegue C, Fassler R (2011) Quantitative proteomics of the integrin adhesome show a myosin II-dependent recruitment of LIM domain proteins. EMBO Rep 12(3):259–266. https://doi.org/10.1038/embor.2011.5

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Kuo JC, Han X, Hsiao CT, Yates JR 3rd, Waterman CM (2011) Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for beta-Pix in negative regulation of focal adhesion maturation. Nat Cell Biol 13(4):383–393. https://doi.org/10.1038/ncb2216

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B (2007) Functional atlas of the integrin adhesome. Nat Cell Biol 9(8):858–867. https://doi.org/10.1038/ncb0807-858

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Horton ER, Byron A, Askari JA, Ng DHJ, Millon-Fremillon A, Robertson J, Koper EJ, Paul NR, Warwood S, Knight D, Humphries JD, Humphries MJ (2015) Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly. Nat Cell Biol 17(12):1577–1587. https://doi.org/10.1038/ncb3257

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Byron A, Humphries JD, Craig SE, Knight D, Humphries MJ (2012) Proteomic analysis of alpha4beta1 integrin adhesion complexes reveals alpha-subunit-dependent protein recruitment. Proteomics 12(13):2107–2114. https://doi.org/10.1002/pmic.201100487

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Robertson J, Jacquemet G, Byron A, Jones MC, Warwood S, Selley JN, Knight D, Humphries JD, Humphries MJ (2015) Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling. Nat Commun 6:6265. https://doi.org/10.1038/ncomms7265

    Article  CAS  PubMed  Google Scholar 

  37. Kechagia JZ, Ivaska J, Roca-Cusachs P (2019) Integrins as biomechanical sensors of the microenvironment. Nat Rev Mol Cell Biol 20(8):457–473. https://doi.org/10.1038/s41580-019-0134-2

    Article  CAS  PubMed  Google Scholar 

  38. Evans EA, Calderwood DA (2007) Forces and bond dynamics in cell adhesion. Science 316(5828):1148–1153. https://doi.org/10.1126/science.1137592

    Article  CAS  PubMed  Google Scholar 

  39. Moore SW, Roca-Cusachs P, Sheetz MP (2010) Stretchy proteins on stretchy substrates: the important elements of integrin-mediated rigidity sensing. Dev Cell 19(2):194–206. https://doi.org/10.1016/j.devcel.2010.07.018

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Sun Z, Costell M, Fassler R (2019) Integrin activation by talin, kindlin and mechanical forces. Nat Cell Biol 21(1):25–31. https://doi.org/10.1038/s41556-018-0234-9

    Article  CAS  PubMed  Google Scholar 

  41. Hoffmann JE, Fermin Y, Stricker RL, Ickstadt K, Zamir E (2014) Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol. Elife 3:e02257. https://doi.org/10.7554/eLife.02257

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Han SJ, Dean KM, Whitewood J, Bachir A, Guttierrez E, Groisman A, Horwitz AR, Goult BT, Danuser G (2019) Formation of talin-vinculin pre-complexes dictates maturation of nascent adhesions by accelerated force transmission and vinculin recruitment. bioRxiv:735183. https://doi.org/10.1101/735183

  43. Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg MH, Calderwood DA (2003) Talin binding to integrin beta tails: a final common step in integrin activation. Science 302(5642):103–106. https://doi.org/10.1126/science.1086652

    Article  CAS  PubMed  Google Scholar 

  44. Woodside DG, Obergfell A, Talapatra A, Calderwood DA, Shattil SJ, Ginsberg MH (2002) The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains. J Biol Chem 277(42):39401–39408. https://doi.org/10.1074/jbc.M207657200

    Article  CAS  PubMed  Google Scholar 

  45. Sungkaworn T, Jobin ML, Burnecki K, Weron A, Lohse MJ, Calebiro D (2017) Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature 550(7677):543–547. https://doi.org/10.1038/nature24264

    Article  CAS  PubMed  Google Scholar 

  46. Chazeau A, Mehidi A, Nair D, Gautier JJ, Leduc C, Chamma I, Kage F, Kechkar A, Thoumine O, Rottner K, Choquet D, Gautreau A, Sibarita JB, Giannone G (2014) Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion. EMBO J 33(23):2745–2764. https://doi.org/10.15252/embj.201488837

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Remorino A, De Beco S, Cayrac F, Di Federico F, Cornilleau G, Gautreau A, Parrini MC, Masson JB, Dahan M, Coppey M (2017) Gradients of Rac1 nanoclusters support spatial patterns of Rac1 signaling. Cell Rep 21(7):1922–1935. https://doi.org/10.1016/j.celrep.2017.10.069

    Article  CAS  PubMed  Google Scholar 

  48. Mehidi A, Rossier O, Schaks M, Chazeau A, Biname F, Remorino A, Coppey M, Karatas Z, Sibarita JB, Rottner K, Moreau V, Giannone G (2019) Transient activations of Rac1 at the lamellipodium tip trigger membrane protrusion. Curr Biol 29(17):2852–2866. e2855. https://doi.org/10.1016/j.cub.2019.07.035

    Article  CAS  PubMed  Google Scholar 

  49. Shibata AC, Chen LH, Nagai R, Ishidate F, Chadda R, Miwa Y, Naruse K, Shirai YM, Fujiwara TK, Kusumi A (2013) Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single-molecule analysis. Cytoskeleton 70(3):161–177. https://doi.org/10.1002/cm.21097

    Article  CAS  PubMed  Google Scholar 

  50. Tsunoyama TA, Watanabe Y, Goto J, Naito K, Kasai RS, Suzuki KGN, Fujiwara TK, Kusumi A (2018) Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function. Nat Chem Biol 14(5):497–506. https://doi.org/10.1038/s41589-018-0032-5

    Article  CAS  PubMed  Google Scholar 

  51. Leduc C, Si S, Gautier J, Soto-Ribeiro M, Wehrle-Haller B, Gautreau A, Giannone G, Cognet L, Lounis B (2013) A highly specific gold nanoprobe for live-cell single-molecule imaging. Nano Lett 13(4):1489–1494. https://doi.org/10.1021/nl304561g

    Article  CAS  PubMed  Google Scholar 

  52. Paszek MJ, DuFort CC, Rossier O, Bainer R, Mouw JK, Godula K, Hudak JE, Lakins JN, Wijekoon AC, Cassereau L, Rubashkin MG, Magbanua MJ, Thorn KS, Davidson MW, Rugo HS, Park JW, Hammer DA, Giannone G, Bertozzi CR, Weaver VM (2014) The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature 511(7509):319–325. https://doi.org/10.1038/nature13535

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM (2010) Nanoscale architecture of integrin-based cell adhesions. Nature 468(7323):580–584. https://doi.org/10.1038/nature09621

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  54. Orre, T. et al. (2020) ‘Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions’, bioRxiv, p. 2020.08.06.239731. doi: 10.1101/2020.08.06.239731

    Google Scholar 

  55. Spiess M, Hernandez-Varas P, Oddone A, Olofsson H, Blom H, Waithe D, Lock JG, Lakadamyali M, Stromblad S (2018) Active and inactive beta1 integrins segregate into distinct nanoclusters in focal adhesions. J Cell Biol 217(6):1929–1940. https://doi.org/10.1083/jcb.201707075

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  56. Rys JP, DuFort CC, Monteiro DA, Baird MA, Oses-Prieto JA, Chand S, Burlingame AL, Davidson MW, Alliston TN (2015) Discrete spatial organization of TGFbeta receptors couples receptor multimerization and signaling to cellular tension. Elife 4:e09300. https://doi.org/10.7554/eLife.09300

    Article  PubMed Central  PubMed  Google Scholar 

  57. Kiosses WB, Shattil SJ, Pampori N, Schwartz MA (2001) Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration. Nat Cell Biol 3(3):316–320. https://doi.org/10.1038/35060120

    Article  CAS  PubMed  Google Scholar 

  58. Del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, Schwartz MA (2002) Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol 4(3):232–239. https://doi.org/10.1038/ncb759

    Article  CAS  PubMed  Google Scholar 

  59. Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440(7087):1069–1072. https://doi.org/10.1038/nature04665

    Article  CAS  PubMed  Google Scholar 

  60. Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn KM (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461(7260):104–108. https://doi.org/10.1038/nature08241

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  61. Yazawa M, Sadaghiani AM, Hsueh B, Dolmetsch RE (2009) Induction of protein-protein interactions in live cells using light. Nat Biotechnol 27(10):941–945. https://doi.org/10.1038/nbt.1569

    Article  CAS  PubMed  Google Scholar 

  62. Valon L, Etoc F, Remorino A, di Pietro F, Morin X, Dahan M, Coppey M (2015) Predictive spatiotemporal manipulation of signaling perturbations using optogenetics. Biophys J 109(9):1785–1797. https://doi.org/10.1016/j.bpj.2015.08.042

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  63. Kasai RS, Suzuki KG, Prossnitz ER, Koyama-Honda I, Nakada C, Fujiwara TK, Kusumi A (2011) Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J Cell Biol 192(3):463–480. https://doi.org/10.1083/jcb.201009128

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  64. Dustin ML, Depoil D (2011) New insights into the T cell synapse from single molecule techniques. Nat Rev Immunol 11(10):672–684. https://doi.org/10.1038/nri3066

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  65. Opazo P, Sainlos M, Choquet D (2012) Regulation of AMPA receptor surface diffusion by PSD-95 slots. Curr Opin Neurobiol 22(3):453–460. https://doi.org/10.1016/j.conb.2011.10.010

    Article  CAS  PubMed  Google Scholar 

  66. Chamma I, Rossier O, Giannone G, Thoumine O, Sainlos M (2017) Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin. Nat Protoc 12(4):748–763. https://doi.org/10.1038/nprot.2017.010

    Article  CAS  PubMed  Google Scholar 

  67. Izeddin I, Boulanger J, Racine V, Specht CG, Kechkar A, Nair D, Triller A, Choquet D, Dahan M, Sibarita JB (2012) Wavelet analysis for single molecule localization microscopy. Opt Express 20(3):2081–2095. https://doi.org/10.1364/OE.20.002081

    Article  CAS  PubMed  Google Scholar 

  68. Racine V, Hertzog A, Jouanneau J, Salamero J, Kervrann C, Sibarita J (2006) Multiple-target tracking of 3D fluorescent objects based on simulated annealing. In: Third IEEE international symposium on biomedical imaging: nano to macro, 6–9 Apr 2006, pp 1020–1023. https://doi.org/10.1109/ISBI.2006.1625094

  69. Racine V, Sachse M, Salamero J, Fraisier V, Trubuil A, Sibarita JB (2007) Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells. J Microsc 225(Pt 3):214–228. https://doi.org/10.1111/j.1365-2818.2007.01723.x

    Article  PubMed  Google Scholar 

  70. Das S, Yin T, Yang Q, Zhang J, Wu YI, Yu J (2015) Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling. Proc Natl Acad Sci U S A 112(3):E267–E276. https://doi.org/10.1073/pnas.1409667112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  71. El Beheiry M, Dahan M, Masson JB (2015) InferenceMAP: mapping of single-molecule dynamics with Bayesian inference. Nat Methods 12(7):594–595. https://doi.org/10.1038/nmeth.3441

    Article  CAS  PubMed  Google Scholar 

  72. De Keersmaecker H, Camacho R, Rantasa DM, Fron E, Uji IH, Mizuno H, Rocha S (2018) Mapping transient protein interactions at the nanoscale in living mammalian cells. ACS Nano 12(10):9842–9854. https://doi.org/10.1021/acsnano.8b01227

    Article  CAS  PubMed  Google Scholar 

  73. Huang F, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, Bewersdorf J (2013) Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat Methods 10(7):653–658. https://doi.org/10.1038/nmeth.2488

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  74. Almada P, Culley S, Henriques R (2015) PALM and STORM: into large fields and high-throughput microscopy with sCMOS detectors. Methods 88:109–121. https://doi.org/10.1016/j.ymeth.2015.06.004

    Article  CAS  PubMed  Google Scholar 

  75. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106(9):3125–3130. https://doi.org/10.1073/pnas.0813131106

    Article  PubMed Central  PubMed  Google Scholar 

  76. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795. https://doi.org/10.1038/nmeth929

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  77. van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6(7):991–1009. https://doi.org/10.1038/nprot.2011.336

    Article  CAS  PubMed  Google Scholar 

  78. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813. https://doi.org/10.1126/science.1153529

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  79. Bourg N, Mayet C, Dupuis G, Barroca T, Bon P, Lécart S, Fort E, Lévêque-Fort S (2015) Direct optical nanoscopy with axially localized detection. Nat Photonics 9(9):587–593. https://doi.org/10.1038/nphoton.2015.132

    Article  CAS  Google Scholar 

  80. Fort E, Grésillon S (2007) Surface enhanced fluorescence. J Phys D Appl Phys 41(1):013001. https://doi.org/10.1088/0022-3727/41/1/013001

    Article  CAS  Google Scholar 

  81. Ruckstuhl T, Rankl M, Seeger S (2003) Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument. Biosens Bioelectron 18(9):1193–1199. https://doi.org/10.1016/s0956-5663(02)00239-7

    Article  CAS  PubMed  Google Scholar 

  82. Deschamps J, Mund M, Ries J (2014) 3D superresolution microscopy by supercritical angle detection. Opt Express 22(23):29081–29091. https://doi.org/10.1364/OE.22.029081

    Article  CAS  PubMed  Google Scholar 

  83. Theodosiou M, Widmaier M, Bottcher RT, Rognoni E, Veelders M, Bharadwaj M, Lambacher A, Austen K, Muller DJ, Zent R, Fassler R (2016) Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin. Elife 5:e10130. https://doi.org/10.7554/eLife.10130

    Article  PubMed Central  PubMed  Google Scholar 

  84. Hirbawi J, Bialkowska K, Bledzka KM, Liu J, Fukuda K, Qin J, Plow EF (2017) The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation. J Biol Chem 292(34):14258–14269. https://doi.org/10.1074/jbc.M117.776195

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  85. Shi X, Ma YQ, Tu Y, Chen K, Wu S, Fukuda K, Qin J, Plow EF, Wu C (2007) The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility. J Biol Chem 282(28):20455–20466. https://doi.org/10.1074/jbc.M611680200

    Article  CAS  PubMed  Google Scholar 

  86. Rossier O, Giannone G (2016) The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking. Exp Cell Res 343(1):28–34. https://doi.org/10.1016/j.yexcr.2015.11.004

    Article  CAS  PubMed  Google Scholar 

  87. Calderwood DA, Campbell ID, Critchley DR (2013) Talins and kindlins: partners in integrin-mediated adhesion. Nat Rev Mol Cell Biol 14(8):503–517. https://doi.org/10.1038/nrm3624

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  88. Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9(9):690–701. https://doi.org/10.1038/nrm2476

    Article  CAS  PubMed  Google Scholar 

  89. Kechkar A, Nair D, Heilemann M, Choquet D, Sibarita JB (2013) Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLoS One 8(4):e62918. https://doi.org/10.1371/journal.pone.0062918

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  90. Ovesny M, Krizek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16):2389–2390. https://doi.org/10.1093/bioinformatics/btu202

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  91. de Chaumont F, Dallongeville S, Chenouard N, Herve N, Pop S, Provoost T, Meas-Yedid V, Pankajakshan P, Lecomte T, Le Montagner Y, Lagache T, Dufour A, Olivo-Marin JC (2012) Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9(7):690–696. https://doi.org/10.1038/nmeth.2075

    Article  CAS  PubMed  Google Scholar 

  92. Chenouard N, Bloch I, Olivo-Marin JC (2013) Multiple hypothesis tracking for cluttered biological image sequences. IEEE Trans Pattern Anal Mach Intell 35(11):2736–3750. https://doi.org/10.1109/TPAMI.2013.97

    Article  PubMed  Google Scholar 

  93. Zala D, Hinckelmann MV, Yu H, Lyra da Cunha MM, Liot G, Cordelieres FP, Marco S, Saudou F (2013) Vesicular glycolysis provides on-board energy for fast axonal transport. Cell 152(3):479–491. https://doi.org/10.1016/j.cell.2012.12.029

    Article  CAS  PubMed  Google Scholar 

  94. Khan AO, Simms VA, Pike JA, Thomas SG, Morgan NV (2017) CRISPR-Cas9 mediated labelling allows for single molecule imaging and resolution. Sci Rep 7(1):8450. https://doi.org/10.1038/s41598-017-08493-x

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  95. Schaks M, Singh SP, Kage F, Thomason P, Klunemann T, Steffen A, Blankenfeldt W, Stradal TE, Insall RH, Rottner K (2018) Distinct interaction sites of Rac GTPase with WAVE regulatory complex have non-redundant functions in vivo. Curr Biol 28(22):3674–3684. e3676. https://doi.org/10.1016/j.cub.2018.10.002

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  96. Pertz O (2010) Spatio-temporal Rho GTPase signaling – where are we now? J Cell Sci 123(Pt 11):1841–1850. https://doi.org/10.1242/jcs.064345

    Article  CAS  PubMed  Google Scholar 

  97. Cherfils J, Zeghouf M (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93(1):269–309. https://doi.org/10.1152/physrev.00003.2012

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France

    A. V. Radhakrishnan, Tianchi Chen, Jose Filipe Nunes Vicente, Thomas Orré, Amine Mehidi, Olivier Rossier & Grégory Giannone

  2. CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France

    A. V. Radhakrishnan, Tianchi Chen, Jose Filipe Nunes Vicente, Thomas Orré, Amine Mehidi, Olivier Rossier & Grégory Giannone

  3. Department of Biochemistry, University of Geneva, Geneva, Switzerland

    Amine Mehidi

Authors
  1. A. V. Radhakrishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tianchi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jose Filipe Nunes Vicente
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Orré
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amine Mehidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olivier Rossier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Grégory Giannone
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Grégory Giannone .

Editor information

Editors and Affiliations

  1. Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientóficas (CSIC)-University of Salamanca, Salamanca, Spain

    Miguel Vicente-Manzanares

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Radhakrishnan, A.V. et al. (2021). Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration. In: Vicente-Manzanares, M. (eds) The Integrin Interactome. Methods in Molecular Biology, vol 2217. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0962-0_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0962-0_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0961-3

  • Online ISBN: 978-1-0716-0962-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation