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Sci Rep. 2018 Jan 25;8(1):1575. doi: 10.1038/s41598-018-20115-8.

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.

Author information

1
Laboratory of Biomechanics, Department of Biosystems Science, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo, Kyoto, 606-8507, Japan.
2
Department of Micro Engineering, Graduate School of Engineering, Kyoto University, Yoshida Honmachi, Sakyo, Kyoto, 606-8501, Japan.
3
National Institute for Nanomaterials Technology, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-Gu, Pohang, Gyeongbuk, 790-784, Korea.
4
Structural Biology Laboratory, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
5
Department of Cell Biology, Graduate School of Medical Science, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, Tokushima, 770-8503, Japan.
6
Laboratory of Biomechanics, Department of Biosystems Science, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo, Kyoto, 606-8507, Japan. adachi@frontier.kyoto-u.ac.jp.
7
Department of Micro Engineering, Graduate School of Engineering, Kyoto University, Yoshida Honmachi, Sakyo, Kyoto, 606-8501, Japan. adachi@frontier.kyoto-u.ac.jp.

Abstract

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.

PMID:
29371682
PMCID:
PMC5785519
DOI:
10.1038/s41598-018-20115-8
[Indexed for MEDLINE]
Free PMC Article

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