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

Abstract

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.

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Acknowledgements

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.

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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.

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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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Videos

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). https://doi.org/10.1038/s41589-018-0032-5

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