Regulation of actin dynamics during myofibril assembly

描述(申请人提供)：横纹肌中的肌节是收缩器官的基本单位。组装和维护有组织的肌节结构对于产生收缩力量是必不可少的。肌动蛋白是肌节细丝的主要成分之一，其长度和方向在横纹肌中受到精确调控。然而，肌节肌动蛋白细丝的组装和维持机制复杂且知之甚少。更令人费解的是，细丝内的肌动蛋白亚基可以动态交换，而不会影响整体结构和收缩功能。在骨骼肌中已经确定了许多肌动蛋白动力学的调节因子，其中一些与遗传性肌肉疾病有关。特别是，线虫性肌病涉及在骨骼肌中形成异常的富含肌动蛋白的聚集体或杆状，由肌动蛋白突变或肌动蛋白动力学调节因子包括星云蛋白、原肌球蛋白和粘连蛋白引起。因此，肌动蛋白动力学的调节对于建立骨骼肌功能收缩装置是至关重要的，而这个系统的故障会导致肌肉功能紊乱。为了研究肌动蛋白在横纹肌中的动态调控机制，我们一直以线虫秀丽线虫为模型系统。线虫的体壁肌肉是横纹肌，大部分肌节蛋白在线虫和人类之间是保守的。对这些肌肉蛋白的强大遗传学研究促进了我们对肌原纤维组装和功能机制的了解。利用这个系统，我们已经确认ADF/cofilin和AIP1促进肌动蛋白细丝的周转，是肌动蛋白细丝有组织组装所必需的，而原肌球蛋白和类钙蛋白则拮抗ADF/cofilin和稳定肌动蛋白细丝。这些发现使我们假设，肌动蛋白动力学的增强子和抑制子之间的平衡对肌节的组装和维持至关重要。最近，我们获得了更多的肌动蛋白动力学调节因子参与肌节肌动蛋白调控的证据。因此，我们建议从三个方面进一步研究它们在肌原纤维组装中的作用：(目的1)我们鉴定了一种富含在体壁肌肉中的环化酶相关蛋白亚型，并将确定环化酶相关蛋白如何与ADF/cofilin和AIP1协同调节肌动蛋白的转换；(目的2)我们鉴定了一种具有很强肌动蛋白切断活性的明胶蛋白样蛋白，该蛋白 (目标3)我们展示了肌节细丝尖端附近不同的肌动蛋白调节蛋白的定位模式，并将研究肌动蛋白动力学在这个专门区域是如何调节的。我们期望这项研究的结果将为肌动蛋白在横纹肌中的动态调控提供新的见解。

DESCRIPTION (provided by applicant): Sarcomere in striate muscle is the basic unit of contractile apparatuses. Assembly and maintenance of organized sarcomeric structure are essential for producing contractile forces. Actin is one of the major components of sarcomeric thin filaments, and length and orientation of the filaments are precisely regulated in striated muscle. However, the mechanism of assembly and maintenance of sarcomeric actin filaments is complex and poorly understood. What is more puzzling is that actin subunits within thin filaments are dynamically exchanged without compromising overall structure and contractile functions. A number of regulators of actin dynamics have been identified in skeletal muscle, and some of them are linked to genetic muscle disorders. In particular, nemaline myopathy involves formation of abnormal actin-rich aggregates or rods in skeletal muscle and is caused by mutations in actin or regulators of actin dynamics including nebulin, tropomyosin, and cofilin. Therefore, the regulation of actin dynamics is fundamentally important for building functional contractile apparatuses in skeletal muscle, and malfunction in this system leads to muscle disorders. To investigate the regulatory mechanism of actin dynamics in striated muscle, we have been using the nematode Caenorhabditis elegans as a model system. Body wall muscle of C. elegans is striated muscle, and most of sarcomeric proteins are conserved between C. elegans and humans. Powerful genetic studies on these muscle proteins advanced our knowledge on the mechanism of myofibril assembly and function. Using this system, we have identified that ADF/cofilin and AIP1 enhance turnover of actin filaments and essential for organized assembly of sarcomeric actin filaments, and that tropomyosin and calponin-like protein antagonize ADF/cofilin and stabilize sarcomeric actin filaments. These findings led us to hypothesize that a balance between enhancers and suppressors of actin dynamics is crucial for sarcomere assembly and maintenance. Recently, we obtained evidence that additional regulators of actin dynamics are involved in sarcomeric actin regulation. Therefore, we propose to further investigate their roles in myofibril assembly in three specific aims: (Aim 1) We identified a cyclase-associated protein isoform that is enriched in body wall muscle and will determine how cyclase-associated protein cooperate with ADF/cofilin and AIP1 to regulate actin turnover, (Aim 2) We identified a gelsolin-like protein with strong actin-severing activity that is enriched in body wall muscle and will investigate its role in actin organization, and (Aim 3) We demonstrated distinct localization patterns of actin-regulatory proteins near the pointed ends of sarcomeric thin filaments and will investigate how actin dynamics are regulated in this specialized region. We expect that results of this research will provide new insight into the regulation of actin dynamics in striated muscle.