Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping.
Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping.
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DOI：
10.1371/journal.pbio.1000387
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发表时间：
2010-06-01
期刊：
影响因子：
9.8
通讯作者：
Scita G
中科院分区：
文献类型：
--
作者：
Hertzog M;Milanesi F;Hazelwood L;Disanza A;Liu H;Perlade E;Malabarba MG;Pasqualato S;Maiolica A;Confalonieri S;Le Clainche C;Offenhauser N;Block J;Rottner K;Di Fiore PP;Carlier MF;Volkmann N;Hanein D;Scita G
肌动蛋白结合蛋白 EPS8 作为肌动蛋白帽和肌动蛋白捆绑因子的不寻常的双重功能映射到该蛋白质的独特结构特征和体内不同的生理活性。肌动蛋白加帽和交联蛋白调节不同细胞突起的动力学和结构。 Eps8 是能够侧向结合和捆绑肌动蛋白丝的独特加帽蛋白家族的创始成员。然而，Eps8 发挥这些功能的结构基础仍然难以捉摸。在这里，我们将生化、分子和遗传学方法与电子显微镜和图像分析相结合，剖析了 Eps8 独特活性的分子机制。我们认为 Eps8 的成束活性主要由紧凑的四螺旋束介导，该四螺旋束沿着丝接触三个肌动蛋白亚基。加帽活性主要由两亲性螺旋介导，该螺旋结合在肌动蛋白倒刺末端的疏水口袋内，阻止肌动蛋白单体的进一步添加。单点诱变验证了这些结合模式，使我们能够从体外捆绑活性中剖析 Eps8 封端。我们进一步表明，Eps8 的加帽和捆绑活性可以在体内完全剖析，证明了已识别的 Eps8 结构/功能模块的生理相关性。 Eps8 通过其加帽活性控制基于肌动蛋白的运动，同时作为捆绑器，对于发育中的秀丽隐杆线虫的适当肠道形态发生至关重要。 细胞骨架的关键成分之一是肌动蛋白，它使细胞能够移动。基于肌动蛋白的运动涉及许多生物过程，例如肠道发育、细胞内运输和细胞迁移。肌动蛋白单体是可以连接在一起形成肌动蛋白丝的独立构件。许多肌动蛋白结合蛋白参与控制细胞内这些肌动蛋白丝的高级结构和动力学。例如，肌动蛋白加帽蛋白通过控制生长的丝末端的数量来调节肌动蛋白动力学，而肌动蛋白交联或捆绑蛋白决定如何将这些丝组织成更高阶的结构。 Eps8 蛋白能够封盖和捆绑肌动蛋白丝。然而，Eps8 这种双重作用的结构基础仍然未知。在这项研究中，我们结合使用多种技术来揭示 Eps8 与肌动蛋白丝相互作用的分子和结构基础。我们表明 Eps8 的不同结构模块负责封盖与捆绑活性，并且我们确定了这些模块在体外和体内的贡献。在功能水平上，我们发现 Eps8 通过其加帽活性调节基于肌动蛋白的运动和细胞运输，而 Eps8 介导的成束对于肠道形态发生至关重要。
The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo. Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans. One of the key components of the cytoskeleton of cells is actin, which allows cells to move. Actin-based motility is involved in many biological processes, such as intestinal development, intracellular trafficking and cell migration. Actin monomers are individual building blocks that can be linked together to form actin filaments. Numerous actin-binding proteins are involved in controlling the higher order architecture and dynamics of these actin filaments within cells. For example, actin capping proteins regulate actin dynamics by controlling the number of growing filament ends, and actin cross-linking or bundling proteins determine how to organize these filaments into higher order structures. The protein Eps8 is capable of capping as well as bundling actin filaments. However, the structural basis of this dual role of Eps8 remains unknown. In this study, we use a combination of techniques to unravel the molecular and structural basis of Eps8 interactions with actin filaments. We show that distinct structural modules of Eps8 are responsible for capping versus bundling activity, and we determine the contributions of these modules in vitro and in vivo. At the functional level, we find that Eps8 regulates actin-based motility and cellular trafficking through its capping activity, whereas Eps8-mediated bundling is essential for intestinal morphogenesis.
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