Regulation of actin dynamics during myofibril assembly

描述(申请人提供)：肌动蛋白细胞骨架适应许多不同的结构，并参与不同的细胞功能。细胞骨架结构的分化通常由许多肌动蛋白结合蛋白控制，这些蛋白调节肌动蛋白细丝的动态和组织。横纹肌中的肌原纤维是肌动蛋白细胞骨架的高度分化形式，专门用于产生收缩力量。然而，肌动蛋白细丝在发育过程中组装成肌原纤维并维持在收缩装置中的机制在很大程度上是未知的。线虫线虫的体壁有倾斜的横纹肌，在生化和功能上与脊椎动物的横纹肌有许多相似之处。我们已经确定肌动蛋白解聚因子(ADF)/cofilin、肌动蛋白相互作用蛋白1(AIP1)和原肌球蛋白是肌动蛋白组织的关键调节因子。当这些蛋白质有缺陷时，肌动蛋白细丝就会紊乱，体壁肌肉的收缩能力就会受损。此外，我们获得的证据表明，类钙蛋白UNC-87和类免疫球蛋白重复蛋白Kettin可能稳定肌动蛋白细丝，而肌肉特异性轮廓蛋白PFN-3可能有助于回收肌动蛋白单体进行聚合。尽管对这些蛋白质的遗传和生化分析表明，这些肌动蛋白结合蛋白中的每一个都可能对肌肉细胞中肌动蛋白的动态调节起重要作用，但我们并不了解这些蛋白质是如何功能相互作用的。我们假设ADF/cofilin是肌动蛋白动力学的关键增强剂，并在功能上与其他肌动蛋白结合蛋白相互作用，调节肌动蛋白的聚合、解聚和在横纹肌原纤维的组装和维护过程中的稳定性。在本项目中，我们旨在研究线虫肌肉细胞中肌动蛋白动力学的三个方面的机制：(目的1)确定ADF/cofilin和AIP1如何分解肌肉细胞中的肌动蛋白细丝；(目的2)确定肌动蛋白单体如何在肌肉细胞中循环用于聚合；以及(目的3)确定肌动蛋白细丝在肌肉细胞中是如何稳定的。这些研究不仅应该为肌原纤维的正常组装和维持机制提供重要的信息，而且还可以为线状肌病的发病机制提供重要的信息，线状肌病与肌动蛋白细丝稳定性的失调有关。

DESCRIPTION (provided by applicant): The actin cytoskeleton is adapted to many different structures and involved in diverse cellular functions. Differentiation of the cytoskeletal structures is generally controlled by a number of actin-binding proteins that regulate dynamics and organization of the actin filaments. Myofibrils in striated muscle are highly differentiated forms of the actin cytoskeleton and specialized for generating contractile forces. However, the mechanisms by which actin filaments are assembled into myofibrils during development and maintained in the contractile apparatuses are largely unknown. The nematode Caenorhabditis elegans has obliquely striated muscle in the body wall, which shares a number of biochemical and functional similarities with vertebrate striated muscle. We have identified actin depolymerizing factor (ADF)/cofilin, actin-interacting protein 1 (AIP1), and tropomyosin as key regulators of actin organization in the body wall muscle. When these proteins are defective, the actin filaments are disorganized and the contractility of the body wall muscle is impaired. In addition, we obtained evidence that UNC-87, a calponin-like protein, and kettin, immunoglobulin-like repeat protein, may stabilize actin filaments, and PFN-3, a muscle-specific profilin, may help recycling actin monomers for polymerization. Although genetic and biochemical analyses of these proteins suggest that each of these actin-binding proteins might be important for regulation of actin dynamics in muscle cells, we do not understand how these proteins functionally interact. We hypothesize that ADF/cofilin is a key enhancer of actin dynamics and functionally interacts with other actin-binding proteins to regulate actin polymerization, depolymerization, and stability during assembly and maintenance of the striated myofibrils. In this project, we aim to investigate mechanisms of three aspects of actin dynamics in muscle cells in C. elegans: (Aim 1) To determine how ADF/cofilin and AIP1 disassemble actin filaments in muscle cells, (Aim 2) To determine how actin monomers are recycled for polymerization in muscle cells, and (Aim 3) To determine how actin filaments are stabilized in muscle cells. These studies should provide important information not only on the mechanism of normal myofibril assembly and maintenance but also on the pathogenesis of nemaline myopathy in which misregulation of actin filament stability is implicated.