Regulation of actin dynamics during myofibril assembly

描述（申请人提供）：横纹肌肌节是收缩装置的基本单位。有组织的肌肉结构的组装和维护对于产生收缩力是必不可少的。肌动蛋白是肌纤维细丝的主要组成成分之一，细丝的长度和方向在横纹肌中受到精确的调控。然而，肌动蛋白细丝的组装和维持机制是复杂的，人们对其了解甚少。更令人困惑的是，细丝内的肌动蛋白亚基在不影响整体结构和收缩功能的情况下动态交换。在骨骼肌中发现了许多肌动蛋白动力学调节因子，其中一些与遗传性肌肉疾病有关。特别是，线状肌病涉及骨骼肌中异常的富含肌动蛋白的聚集体或棒的形成，并由肌动蛋白或肌动蛋白动力学调节因子（包括肌动蛋白、原肌球蛋白和cofilin）的突变引起。因此，肌动蛋白动力学的调节对于在骨骼肌中建立功能性收缩装置至关重要，而该系统的功能障碍会导致肌肉疾病。为了研究肌动蛋白动力学在横纹肌中的调控机制，我们以秀丽隐杆线虫为模型系统。秀丽隐杆线虫的体壁肌为横纹肌，大部分肌合成蛋白在秀丽隐杆线虫和人之间是保守的。对这些肌肉蛋白的强有力的遗传研究促进了我们对肌原纤维组装和功能机制的认识。利用该系统，我们发现ADF/cofilin和AIP1促进肌动蛋白丝的周转，对肌动蛋白丝的有组织组装至关重要，原肌球蛋白和钙钙蛋白样蛋白拮抗ADF/cofilin并稳定肌动蛋白丝。这些发现使我们假设肌动蛋白动力学的增强因子和抑制因子之间的平衡对肌节的组装和维持至关重要。最近，我们获得的证据表明，肌动蛋白动力学的其他调节因子参与肌动蛋白调节。因此，我们提出在三个特定目标中进一步研究它们在肌原纤维组装中的作用：（目的1）我们鉴定了富含体壁肌肉的环化酶相关蛋白异构体，并将确定环化酶相关蛋白如何与ADF/cofilin和AIP1合作调节肌动蛋白周转；（目的2）我们鉴定了具有强肌动蛋白切断活性的凝胶样蛋白

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.