Integrin-bound talin head inhibits actin filament barbed-end elongation
Integrin-bound talin head inhibits actin filament barbed-end elongation
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DOI：
10.1074/jbc.m117.808204
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发表时间：
2018-02-16
影响因子：
4.8
通讯作者：
Le Clainche, Christophe
中科院分区：
文献类型：
--
作者：
Ciobanasu, Corina;Wang, Hong;Le Clainche, Christophe
粘着斑（FA）通过跨膜整合素和肌动蛋白结合蛋白将细胞外基质与动态肌动蛋白细胞骨架机械偶联。蛋白质机械控制力传递沿着该分子轴的分子机制（即调节整联蛋白活化和控制肌动蛋白聚合）在很大程度上仍然未知。Talin是一种主要的肌动蛋白结合蛋白，它控制整联蛋白的由内而外激活和肌动蛋白丝锚定，因此在肌动蛋白-细胞外基质机械偶联的建立中起主要作用。Talin含有三个肌动蛋白结合结构域（ABD）。N-末端头部结构域包含F3整合素激活结构域和ABD 1，而C-末端杆包含肌动蛋白锚定ABD 2和ABD 3。整联蛋白结合受N-末端头部和C-末端五螺旋束（R9）之间的分子内相互作用调节。无论是talin ABDs调节肌动蛋白聚合的组成或调节方式尚未得到充分探讨。在这里，我们结合联合收割机动力学分析，使用荧光光谱和单肌动蛋白纤维的观察，在全内反射荧光显微镜，检查相关功能的三个ABD塔林。我们发现，N-末端ABD 1块肌动蛋白丝倒刺末端伸长，而ABD 2和ABD 3不显示任何活动。通过突变ABD 1中的残基，我们发现这种活性是由带正电的表面介导的，该表面部分被其与R9的分子内相互作用所掩盖。我们的研究结果还表明，一旦这种分子内相互作用被释放，整合素结合的塔林头部保留的能力，抑制肌动蛋白组装。
Focal adhesions (FAs) mechanically couple the extracellular matrix to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis (i.e. modulating integrin activation and controlling actin polymerization) remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin filament anchoring and thus plays a major role in the establishment of the actin-extracellular matrix mechanical coupling. Talin contains three actin-binding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, whereas the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a C-terminal five-helix bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in total internal reflection fluorescence microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed-end elongation, whereas ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, the integrin-bound talin head retains the ability to inhibit actin assembly.