Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.
Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.
复制标题
实时TIRF观察AFM对拉伸的α-catenin募集的Vinculin募集。
DOI：
10.1038/s41598-018-20115-8
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发表时间：
2018-01-25
影响因子：
4.6
通讯作者：
Adachi T
中科院分区：
文献类型：
--
作者：
Maki K;Han SW;Hirano Y;Yonemura S;Hakoshima T;Adachi T
粘附连接 (AJ) 会根据细胞间张力适应性地改变其强度；因此，它们整合单个细胞产生的张力来驱动多细胞动力学，例如胚胎的形态发生变化。在细胞间张力下，α-连环蛋白（AJ 的组成蛋白）充当机械化学传感器，招募纽蛋白以促进肌动蛋白重塑。尽管体内和体外研究表明α-连环蛋白介导的机械转导是一个动态分子过程，涉及张力下α-连环蛋白的构象变化以暴露隐秘的纽蛋白结合位点，但目前还没有合适的实验方法来直接探索该过程。因此，在本研究中，我们结合原子力显微镜（AFM）和全内反射荧光（TIRF）开发了一种新颖的系统。在该系统中，在盖玻片上修饰的 α-连环蛋白分子（残基 276-634；机械敏感的 M1-M3 结构域）通过 AFM 进行拉伸，并使用 TIRF 同时实时观察溶解在溶液中的 Alexa 标记的全长纽蛋白分子的募集。我们对 α-连环蛋白应用了生理上可能的张力和延伸范围，并直接观察了其纽蛋白的募集。我们的新系统可用于机械生物学和生物物理学领域，通过耦合生物力学和生化信息来探索张力下蛋白质的功能。
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.
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DOI：
10.1038/nrm3112
发表时间：
2011-05
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
64.8
作者：
Cavey, Matthieu;Rauzi, Matteo;Lecuit, Thomas
通讯作者：
Lecuit, Thomas
影响因子：
4.8
作者：
Cohen, DM;Chen, H;Craig, SW
通讯作者：
Craig, SW
影响因子：
64.8
作者：
FINER, JT;SIMMONS, RM;SPUDICH, JA
通讯作者：
SPUDICH, JA
影响因子：
38.3
作者：
Kufer, Stefan K.;Strackharn, Mathias;Gaub, Hermann E.
通讯作者：
Gaub, Hermann E.