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Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function


Single-fluorescent-molecule imaging tracking (SMT) is becoming an important tool to study living cells. However, photobleaching and photoblinking (hereafter referred to as photobleaching/photoblinking) of the probe molecules strongly hamper SMT studies of living cells, making it difficult to observe in vivo molecular events and to evaluate their lifetimes (e.g., off rates). The methods used to suppress photobleaching/photoblinking in vitro are difficult to apply to living cells because of their toxicities. Here using 13 organic fluorophores we found that, by combining low concentrations of dissolved oxygen with a reducing-plus-oxidizing system, photobleaching/photoblinking could be strongly suppressed with only minor effects on cells, which enabled SMT for as long as 12,000 frames (~7 min at video rate, as compared to the general 10-s-order durations) with ~22-nm single-molecule localization precisions. SMT of integrins revealed that they underwent temporary (<80-s) immobilizations within the focal adhesion region, which were responsible for the mechanical linkage of the actin cytoskeleton to the extracellular matrix.

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Fig. 1: Photobleaching lifetimes in single-fluorescent-molecule imaging are prolonged under the conditions of low O2+ROXS.
Fig. 2: Photoblinking, in addition to photobleaching, can be suppressed under the optimal conditions of low oxygen concentrations+ROXS, as observed by single-fluorescent-molecule tracking at video rate.
Fig. 3: No cytotoxicity under 2%O2, slight toxicity after ROXS addition, in marked contrast to the toxicity of 0%O2.
Fig. 4: Both β1 and β3 integrin undergo repeated TALLs, often lasting for 43 and 79 s, respectively, due to binding both the extracellular matrix and actin filaments.
Fig. 5: Longer TALLs of β1 and β3 integrin both occurred most frequently where the traction force was proposed to be largest, whereas in the course of the generation and disintegration of the FAs β1 integrin arrived at and departed from the FA, respectively, earlier than β3 integrin.


  1. 1.

    Altman, R. B. et al. Cyanine fluorophore derivatives with enhanced photostability. Nat. Methods 9, 68–71 (2011).

    Article  Google Scholar 

  2. 2.

    Altman, R. B. et al. Enhanced photostability of cyanine fluorophores across the visible spectrum. Nat. Methods 9, 428–429 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  Google Scholar 

  4. 4.

    van der Velde, J. H. M. et al. Mechanism of intramolecular photostabilization in self-healing cyanine fluorophores. ChemPhysChem 14, 4084–4093 (2013).

    Article  Google Scholar 

  5. 5.

    Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015). 3, 250.

    CAS  Article  Google Scholar 

  6. 6.

    van der Velde, J. H. M. et al. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization. Nat. Commun. 7, 10144 (2016).

    Article  Google Scholar 

  7. 7.

    Giloh, H. & Sedat, J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252–1255 (1982).

    CAS  Article  Google Scholar 

  8. 8.

    Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    CAS  Article  Google Scholar 

  9. 9.

    Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Edn Engl. 47, 5465–5469 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Kasper, R. et al. Single-molecule STED microscopy with photostable organic fluorophores. Small 6, 1379–1384 (2010).

    CAS  Article  Google Scholar 

  11. 11.

    Cordes, T., Vogelsang, J. & Tinnefeld, P. On the mechanism of trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    CAS  Article  Google Scholar 

  12. 12.

    Packer, L. & Fuehr, K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267, 423–425 (1977).

    CAS  Article  Google Scholar 

  13. 13.

    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 

  14. 14.

    Edwald, E., Stone, M. B., Gray, E. M., Wu, J. & Veatch, S. L. Oxygen depletion speeds and simplifies diffusion in HeLa cells. Biophys. J. 107, 1873–1884 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Kusumi, A. et al. Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model. Annu. Rev. Cell Dev. Biol. 28, 215–250 (2012).

    CAS  Article  Google Scholar 

  16. 16.

    Mohyeldin, A., Garzón-Muvdi, T. & Quiñones-Hinojosa, A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 7, 150–161 (2010).

    CAS  Article  Google Scholar 

  17. 17.

    Semenza, G. L. Oxygen sensing, hypoxia-inducible factors and disease pathophysiology. Annu. Rev. Pathol. 9, 47–71 (2014).

    CAS  Article  Google Scholar 

  18. 18.

    Sakadzić, S. et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nat. Methods 7, 755–759 (2010).

    Article  Google Scholar 

  19. 19.

    Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C. & Kieda, C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J. Cell. Mol. Med. 15, 1239–1253 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Spencer, J. A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Alejo, J. L., Blanchard, S. C. & Andersen, O. S. Small-molecule photostabilizing agents are modifiers of lipid bilayer properties. Biophys. J. 104, 2410–2418 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Scales, T. M. E. & Parsons, M. Spatial and temporal regulation of integrin signaling during cell migration. Curr. Opin. Cell Biol. 23, 562–568 (2011).

    CAS  Article  Google Scholar 

  23. 23.

    Hirata, H., Tatsumi, H., Hayakawa, K. & Sokabe, M. Non-channel mechanosensors working at focal-adhesion–stress-fiber complex. Pflugers Arch. 467, 141–155 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Leduc, C. et al. A highly specific gold nanoprobe for live-cell single-molecule imaging. Nano Lett. 13, 1489–1494 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Mould, A. P. et al. Integrin activation involves a conformational change in the α1 helix of the β-subunit A-domain. J. Biol. Chem. 277, 19800–19805 (2002).

    CAS  Article  Google Scholar 

  26. 26.

    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 

  27. 27.

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

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 29.

    Oakes, P. W., Beckham, Y., Stricker, J. & Gardel, M. L. Tension is required but not sufficient for focal adhesion maturation without a stress fiber template. J. Cell Biol. 196, 363–374 (2012).

    CAS  Article  Google Scholar 

  30. 30.

    Kong, F., García, 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 

  31. 31.

    Kong, F. et al. Cyclic mechanical reinforcement of integrin–ligand interactions. Mol. Cell 49, 1060–1068 (2013).

    CAS  Article  Google Scholar 

  32. 32.

    Livne, A. & Geiger, B. The inner workings of stress fibers—from contractile machinery to focal adhesions and back. J. Cell Sci. 129, 1293–1304 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Cluzel, C. et al. The mechanisms and dynamics of αvβ3 integrin clustering in living cells. J. Cell Biol. 171, 383–392 (2005).

    CAS  Article  Google Scholar 

  34. 34.

    Wehrle-Haller, B. Assembly and disassembly of cell matrix adhesions. Curr. Opin. Cell Biol. 24, 569–581 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Li, J. & Springer, T. A. Integrin extension enables ultrasensitive regulation by cytoskeletal force. Proc. Natl Acad. Sci. USA 114, 4685–4690 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 14, 1057–1067 (2012).

    CAS  Article  Google Scholar 

  37. 37.

    Wang, Y. & Wang, X. Integrins outside focal adhesions transmit tensions during stable cell adhesion. Sci. Rep. 6, 36959 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    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 

  39. 39.

    Plotnikov, S. V., Pasapera, A. M., Sabass, B. & Waterman, C. M. Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151, 1513–1527 (2012).

    CAS  Article  Google Scholar 

  40. 40.

    Blakely, B. L. et al. A DNA-based molecular probe for optically reporting cellular traction forces. Nat. Methods 11, 1229–1232 (2014).

    CAS  Article  Google Scholar 

  41. 41.

    Morimatsu, M., Mekhdjian, A. H., Chang, A. C., Tan, S. J. & Dunn, A. R. Visualizing the interior architecture of focal adhesions with high-resolution traction maps. Nano Lett. 15, 2220–2228 (2015).

    CAS  Article  Google Scholar 

  42. 42.

    Roca-Cusachs, P., Gauthier, N. C., Del Rio, A. & Sheetz, M. P. Clustering of α5β1 integrins determines adhesion strength, whereas αvβ3 and talin enable mechanotransduction. Proc. Natl Acad. Sci. USA 106, 16245–16250 (2009).

    CAS  Article  Google Scholar 

  43. 43.

    Schiller, H. B. et al. β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments. Nat. Cell Biol. 15, 625–636 (2013).

    CAS  Article  Google Scholar 

  44. 44.

    Wehrle-Haller, B. Structure and function of focal adhesions. Curr. Opin. Cell Biol. 24, 116–124 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Doyle, A. D. & Yamada, K. M. Mechanosensing via cell-matrix adhesions in 3D microenvironments. Exp. Cell Res. 343, 60–66 (2016).

    CAS  Article  Google Scholar 

  46. 46.

    Naruse, K., Sai, X., Yokoyama, N. & Sokabe, M. Uniaxial cyclic stretch induces c-SRC activation and translocation in human endothelial cells via SA channel activation. FEBS Lett. 441, 111–115 (1998).

    CAS  Article  Google Scholar 

  47. 47.

    Ihara, M. et al. Association of the cytoskeletal GTP-binding protein Sept4 (H5) with cytoplasmic inclusions found in Parkinson’s disease and other synucleinopathies. J. Biol. Chem. 278, 24095–24102 (2003).

    CAS  Article  Google Scholar 

  48. 48.

    Lindberg, F. P., Gresham, H. D., Schwarz, E. & Brown, E. J. Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane-spanning domains implicated in αvβ3-dependent ligand binding. J. Cell Biol. 123, 485–496 (1993).

    CAS  Article  Google Scholar 

  49. 49.

    Tsuruta, D. et al. Microfilament-dependent movement of the β3-integrin subunit within focal contacts of endothelial cells. FASEB J. 16, 866–868 (2002).

    CAS  Article  Google Scholar 

  50. 50.

    Koyama-Honda, I. et al. Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. Biophys. J. 88, 2126–2136 (2005).

    CAS  Article  Google Scholar 

  51. 51.

    Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K. & Kusumi, A. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 1071–1081 (2002).

    CAS  Article  Google Scholar 

  52. 52.

    Sahl, S. J., Leutenegger, M., Hilbert, M., Hell, S. W. & Eggeling, C. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc. Natl Acad. Sci. USA 107, 6829–6834 (2010).

    CAS  Article  Google Scholar 

  53. 53.

    Shibata, A. C. E. et al. Archipelago architecture of the focal adhesion: membrane molecules freely enter and exit from the focal adhesion zone. Cytoskeleton 69, 380–392 (2012).

    CAS  Article  Google Scholar 

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We thank M. Sokabe (Nagoya University) for the T24 cell lines, M. Kinoshita (Nagoya University) for the NIH3T3 cell lines, M. Humphries (University of Manchester) for the Itgb1-knockout MEFs, E. Brown (Genentech) for the human CD47 cDNA, J. C. Jones (Northwestern University) for the ITGB3 cDNA, Y. Miwa (Tsukuba University) for the pOStet15T3 vector, T. Goto for the help in some experiments and all members of the Kusumi laboratory for valuable discussions. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (DC1 to T.A.T. (2162), Kiban B to K.G.N.S. (15H04351), Kiban B to T.K.F. (16H04775), and Kiban A and Kiban S to A.K. (24247029 and 16H06386, respectively)), Grants-in-Aid for Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.K.F. (15H01212), and a grant from the Core Research for Evolutional Science and Technology (CREST) project of ‘Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics’ of the Japan Science and Technology Agency (JST) to A.K. (JPMJCR14W2). WPI-iCeMS of Kyoto University is supported by the World Premiere Research Center Initiative (WPI) of the MEXT.

Author information




T.A.T. performed a large majority of the single-fluorescent-molecule tracking experiments and prepared trolox; Y.W., J.G. and K.N. performed some of the single-fluorescent-molecule tracking experiments to examine the effects of O2 and ROXS on the photobleaching of fluorescent dye molecules, under the guidance of T.A.T.; T.K.F. and A.K. developed the single-molecule imaging camera system, set up the single-molecule instruments and developed the analysis software; R.S.K., K.G.N.S., T.K.F. and A.K. participated in extensive discussions during the course of this research; T.A.T., T.K.F. and A.K. conceived and formulated this project, and evaluated and discussed data; T.A.T. and A.K. wrote the manuscript; and all of the authors participated in revising the manuscript.

Corresponding author

Correspondence to Akihiro Kusumi.

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Supplementary Text and Figures

Supplementary Tables 1–5, Supplementary Figures 1–47 and Supplementary Notes 1 and 2

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Supplementary Video 1

Photobleaching of single TMR molecules attached to Halo-CD47 on the live cell surface: control versus 2%O2 + TX. Recorded at video rate (30 Hz) and replayed at 4× real time (raw data for Fig. 1a, top).

Supplementary Video 2

Photobleaching of single ST647 molecules attached to ACP-CD47 on the live cell surface: control versus 2%O2 + TQ20. Recorded at video rate (30 Hz) and replayed at 4× real time (raw data for Fig. 1a, bottom).

Supplementary Video 3

Photobleaching of single Cy5-TX molecules attached to Halo-CD47 on the live cell surface under control, 2%O2 and 0%O2 conditions. Recorded at video rate (30 Hz) and replayed at 4× real time (raw data for Supplementary Fig. 18a, bottom).

Supplementary Video 4

Single molecules of ST647-labeled ACP–integrin β1 (green spots) moved in and out of several FAs (blue binarized images of mGFP-paxillin), exhibiting alternating periods of TALL and thermal diffusion, both inside and outside the FAs (raw data for Fig. 4a, left), under 2%O2 + TQ20. A trajectory is overlaid for one of the ACP-integrin β1 molecules.

Supplementary Video 5

Single molecules of ST647-labeled ACP–integrin β3, exhibiting intermittent TALLs similar to those of ACP–integrin β1 (raw data for Fig. 4a, right).

Supplementary Video 6

Time course of FA diminution (visualized by mGFP-paxillin with color coding) after the addition of 10 µM ROCK inhibitor Y26732 (see Supplementary Fig. 32a for the color scale).

Supplementary Video 7

Time-lapse observation of FA formation and its termination near the cell periphery.

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Tsunoyama, T.A., Watanabe, Y., Goto, J. et al. Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function. Nat Chem Biol 14, 497–506 (2018).

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