Regulation of the actin filament pointed end dynamics in health and disease

参与心肌/骨骼肌收缩的细丝的变化通常会导致 有致命后果的心肌病/线虫性肌病。我们的长期目标是确定 肌肉正常发育过程中肌动蛋白结构调控的组成和分子机制 和疾病。我们的短期目标是确定细丝长度是如何通过 肌动蛋白细丝尖端结合蛋白，leiomodin(Lmod)和troomoduin(Tmod)。聚合温度为 尖端决定细丝的长度，并直接受原调节蛋白(Tmod)的结合调节 和来洛莫定(Lmod)。这种结合被整合的细丝成分原肌球蛋白(TPM)增强。 我们推测，维持细丝长度需要Tmod和Lmod的拮抗作用； 也就是说，Tmod的作用是防止尖端的伸长，而Lmod允许伸长。我们也 预测Tmod和Lmod在指端的结合和作用由不同的排列决定 它们的TPM和肌动蛋白结合部位。为了实现我们的目标，一个强大的多学科合作 在华盛顿州立大学的克斯特尤科娃实验室(拥有蛋白质专业知识)之间建立 肌动蛋白细丝和调节蛋白的结构、结构生化和生物物理性质)和 亚利桑那大学Gregorio实验室(拥有分子、细胞和发育方面的专业知识 肌原纤维组装的生物学)。在这项提案中，我们将结合广泛的最先进的方法 例如确定Lmod/TPM结合界面高分辨率原子结构，以及使用先进的 Lmod2或Lmod3缺失小鼠心肌细胞的显微镜和生理学评估以测试我们的分子 设计。拟议的实验将把LMOD相关的细丝变化与家族性 肌病。更好地理解细丝的功能及其调节对于更好地理解 肌肉疾病的发病机制，以改善诊断，并潜在地确定新的药物靶点。

Alterations in the thin filaments involved in cardiac/skeletal muscle contraction often produce cardiomyopathies/nemaline myopathies with fatal consequences. Our long-term goal is to identify the components and molecular mechanisms regulating actin architecture in muscle during normal development and disease. Our short-term goal is to determine the mechanisms of how thin filament lengths are regulated by the actin filament pointed end binding proteins, leiomodin (Lmod) and tropomoduin (Tmod). Polymerization at the pointed end determines thin filament length and is regulated directly by the binding of tropomodulin (Tmod) and leiomodin (Lmod). This binding is enhanced by the integral thin filament component, tropomyosin (Tpm). We hypothesize that maintenance of thin filament length requires the antagonistic action of Tmod and Lmod; that is, the role of Tmod is to prevent elongation at the pointed end while Lmod allows elongation. We also predict that Tmod and Lmod binding and action at the pointed end is determined by different arrangements of their Tpm- and actin-binding sites. To achieve our goals, a powerful, multidisciplinary collaboration has been established between the Kostyukova laboratory at Washington State University (with expertise in protein structure, structural biochemistry and biophysical properties of actin filaments and regulatory proteins) and the Gregorio laboratory at the University of Arizona (with expertise in the molecular, cellular and developmental biology of myofibril assembly). In this proposal we will combine a broad range of state-of-the-art approaches such as determination of high-resolution atomic structure of Lmod /Tpm binding interface, and use of advanced microscopy and physiological assessment of myocytes from Lmod2 or Lmod3 null mice to test our molecular designs. The proposed experiments will connect Lmod-related thin filament alterations with familial myopathies. A better understanding of thin filament function and of its regulation is critical to better understand muscle disease pathogenesis, to improve diagnostics and to potentially identify novel drug targets.