Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.
Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.
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DOI：
10.1371/journal.pone.0122886
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Yamada S
中科院分区：
文献类型：
--
作者：
Ueda S;Blee AM;Macway KG;Renner DJ;Yamada S
组织和器官受到持续的物理扰动，单个细胞必须对机械力作出反应以维持组织的完整性。然而，机械转导背后的分子相互作用在细胞-细胞连接处并没有完全定义。这部分是由于在力诱导的蛋白质复合物中可能普遍存在的微弱和短暂的相互作用。通过标记α-catenin的混杂生物素连接酶BirA和底物拉伸细胞室的原位近端生物素化，我们试图确定围绕α-catenin的力依赖分子相互作用，α-catenin是一种肌动蛋白调节剂，位于钙粘蛋白介导的细胞-细胞粘附位点。在免疫荧光染色中，E-cadherin、β-catenin、vinculin和actin在细胞-细胞接触处与α-catenin定位，但只有β-catenin和血小板红蛋白被生物素化，这表明这种近端生物素化仅限于紧邻α-catenin的分子。在机械拉伸的样品中，非肌球蛋白IIA的生物素化增加，而不是肌球蛋白IIB，这表明在底物拉伸过程中α-连环蛋白和肌球蛋白IIA在空间上接近。这种力诱导的生物素化作用随着肌球蛋白II活性被blebbistatin抑制而减弱。总之，这项有前途的技术使我们能够识别力敏感复合物，这可能是力承载细胞粘附的机械响应所必需的。
Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.
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影响因子：
64.5
作者：
Drees, F;Pokutta, S;Weis, WI
通讯作者：
Weis, WI
DOI：
10.1083/jcb.201001149
发表时间：
2010-06-28
期刊：
The Journal of cell biology
影响因子：
--
作者：
le Duc Q;Shi Q;Blonk I;Sonnenberg A;Wang N;Leckband D;de Rooij J
通讯作者：
de Rooij J
DOI：
10.1083/jcb.201112098
发表时间：
2012-03-19
期刊：
The Journal of cell biology
影响因子：
--
作者：
Roux KJ;Kim DI;Raida M;Burke B
通讯作者：
Burke B
影响因子：
3.7
作者：
Miyake, Yuka;Inoue, Naoko;Yonemura, Shigenobu
通讯作者：
Yonemura, Shigenobu
影响因子：
7.2
作者：
Meng, Wenxiang;Takeichi, Masatoshi
通讯作者：
Takeichi, Masatoshi