Structure-Function of the Cytoskeleton

本项目研究血管平滑肌肌动蛋白细胞骨架重塑的分子基础 收缩。它补充了该赠款的其他项目，重点是组织，细胞和大分子 组件.两个肌动蛋白结合蛋白家族，Ena/VASP和WASP，已经成为关键的调节剂 的细胞骨架重塑，发挥不同的作用，分别在长丝成核和伸长。的 控制这两个过程的分子机制仍然是一个谜。Ena/VASP和WASP在功能上 不同但共享相似的模块化结构。它们都含有多聚脯氨酸区域，可以介导 前纤维蛋白-肌动蛋白，接着是WASP-同源2（WH 2）型的G-肌动蛋白结合结构域。该项目构建 根据重要的初步结果（在这里介绍）和我们最近确定的各种WH 2- 肌动蛋白复合物，提出以下两个假设：1）串联WH 2排列肌动蛋白亚基沿着 形成用于肌动蛋白组装的核的丝股（成核步骤）。2)poly-Pro-WH 2模块有助于 通过“加工”前纤维蛋白-肌动蛋白复合物使其结合到纤维的有刺末端上， 生长细丝（伸长步骤）。为了检验这些假设，目标1和2将剖析结构- poly-Pro-WH 2和tandem-WH 2模块的功能。我们将确定肌动蛋白的晶体结构 通过串联WH 2杂交构建体组装的微丝和与profilin-actin结合的poly-Pro-WH 2的微丝 （伸长中的过渡状态）。补充解决方案研究将使用分析 超离心和SAXS/WAXS。对各种蛋白质-蛋白质相互作用的生物物理研究 在成核和伸长中的作用将研究变构效应在这些过程中的作用。Aim 3研究 肌动蛋白结合和α-辅肌动蛋白的支架功能，α-辅肌动蛋白是血影蛋白家族的关键衔接蛋白。 α-辅肌动蛋白介导质膜上整合素之间的相互作用和粘着斑 蛋白质zyxin、黏着斑蛋白和paladin，其又将Ena/VASP募集到致密斑块。作为一个范例， 这些相互作用，我们将研究α-辅肌动蛋白-zyxin相互作用的结构基础。使用 SAXS/WAXS，FRET和Au，我们将测试两个流行的F-肌动蛋白结合模型，紧凑和扩展， 对于血影蛋白家族成员的肌动蛋白结合结构域。了解的分子基础 血管肌肉收缩将加速发现治疗心血管疾病的疗法。

This project studies the molecular basis for actin cytoskeleton remodeling in vascular smooth muscle contraction. It complements the other projects of this grants that focus on tissues, cells and macromolecular assemblies. Two families of actin-binding proteins, Ena/VASP and WASP, have emerged as key regulators of cytoskeleton remodeling, playing distinct roles in filament nucleation and elongation, respectively. The molecular mechanisms controlling both processes remain a mystery. Ena/VASP and WASP are functionally distinct but share similar modular structures. They both contain poly-Pro regions that mediate the binding of profilin-actin, followed by G-actin binding domains of the WASP-Homology 2 (WH2) type. This project builds upon important preliminary results (presented here) and our recently determined structures of various WH2- actin complexes to propose the following two hypotheses: 1) Tandem WH2s line up actin subunits along a filament strand forming nuclei for actin assembly (nucleation step). 2) The poly-Pro-WH2 module contributes to filament elongation by "processing" profilin-actin complexes for their incorporation onto the barbed end of growing filaments (elongation step). To test these hypotheses, aims 1 and 2 will dissect the structure- function of the poly-Pro-WH2 and tandem-WH2 modules. We will determine the crystal structures of actin minifilaments assembled via tandem WH2 hybrid constructs and that of poly-Pro-WH2 bound to profilin-actin (transition state in elongation). Complementary solution studies will be carried out using analytical ultracentrifugation and SAXS/WAXS. A biophysical study of the various protein-protein interactions involved in nucleation and elongation will investigate the role of allosteric effects in these processes. Aim 3 studies the actin binding and scaffolding functions of alpha-actinin, a key adaptor protein of the spectrin family. Alpha-actinin mediates the interactions between integrins at the plasma membrane and the focal-adhesion proteins zyxin, vinculin, and paladin, which in turn recruit Ena/VASP to dense plaques. As a paradigm of these interactions, we will study the structural basis for the alpha-actinin-zyxin interaction. Using SAXS/WAXS, FRET and AU, we will test the two prevailing F-actin-binding models, compact and extended, for the actin-binding domain of members of the spectrin family. Understanding the molecular basis of vascular muscle contraction will accelerate the discovery of therapies to treat cardiovascular diseases.