Deciphering how a human mutation in leiomodin-3 leads to muscle disease

项目总结 横纹肌细胞收缩依赖于肌球蛋白(粗)丝和肌动蛋白的适当重叠 (细的)细丝。Leiomodin(Lmod)和Tmod(Tmod)是与薄的尖端结合的蛋白质 细丝，以便微调其长度。Tmod1和Lmod2是心肌中的主要亚型。Lmod3 是主要的骨骼亚型，但它也在心肌中表达。Tmod和Lmod的突变 已被证明会导致细丝长度失调，并导致肌病的发生。目标是 这一建议的目的是确定Tmod和Lmod蛋白如何调节细丝的分子机制。 集合。我们计划通过研究Lmod3中已发现的突变来创建一种新的疾病模型 在患有线状肌病(一种骨骼肌疾病)的患者中。我们假设引入这种突变 在Tmod和Lmod中，会导致细丝长度改变和肌动蛋白组装紊乱，从而导致疾病 发展。我们已经获得了Lmod3基因敲除小鼠模型，它将作为这方面的重要工具 学习。我们提出了以下目标：目标1研究Lmod3在横纹肌中的作用。 Lmod3基因敲除(KO)小鼠品系。将使用免疫荧光去卷积显微镜来评估总体 这些KO小鼠的肌节结构和细丝长度的变化。个体骨骼的收缩力 并对心肌细胞进行测量。我们将尝试预防这些小鼠的骨骼和心脏缺陷 通过腺相关病毒导入Lmod3。目标2将确定线状肌病相关的效果 细丝长度突变和肌动蛋白动力学。突变的Lmod和Tmod蛋白将在 通过腺病毒感染骨骼肌和心肌细胞。细丝将被视觉化，并使用 免疫荧光显微镜，而光漂白后的荧光恢复将检测突变的Lmod和 Tmod组装到尖端的能力。目标3是确定线虫肌病相关突变是如何 影响Lmod/Tmod的结构和功能。将使用圆二色谱来研究突变的能力 Lmod和Tmod正确折叠，并将使用核磁共振来确定结构 改变可能潜在地影响突变的Tmod和Lmod与其他蛋白质的结合界面，如肌动蛋白 和原肌球蛋白。我们将通过执行芘-肌动蛋白来评估突变的Lmod和Tmod的功能变化 聚合试验和共沉淀试验。这个多学科项目的长期目标是， 从单个分子到整个动物的研究，是为了确定肌动蛋白细丝是如何扰动的 长度过长会导致肌肉疾病。这一点很重要，因为肌动蛋白是大多数细胞类型中含量最丰富的蛋白质 并参与了许多重要的细胞过程。这个项目取得的成果将使我们能够 破译细丝长度和肌肉功能之间的联系，以及体内的机械信息 Lmod3的单一突变是如何导致人类肌病的。

PROJECT SUMMARY Striated muscle cell contraction is dependent on the proper overlap of myosin (thick) filaments and actin (thin) filaments. Leiomodin (Lmod) and tropomodulin (Tmod) are proteins that bind to the pointed end of thin filaments in order to fine-tune their lengths. Tmod1 and Lmod2 are the major isoforms in cardiac muscle. Lmod3 is the major skeletal isoform, however it is also expressed in cardiac muscle. Mutations in Tmod and Lmod have been shown to result in dysregulated thin filament lengths and lead to the development of myopathies. The goal of this proposal is to identify molecular mechanisms for how Tmod and Lmod proteins regulate thin filament assembly. We plan to create a novel model of disease by studying a mutation that has been identified in Lmod3 in patients with nemaline myopathy (a skeletal muscle disorder). We hypothesize that introducing this mutation in Tmod and Lmod will result in altered thin filament lengths and perturbed actin assembly, leading to disease development. We have obtained a Lmod3 knockout mouse model, which will serve as an important tool for this study. We propose the following aims: Aim 1 is to examine the role of Lmod3 in striated muscle by utilizing a Lmod3 knockout (KO) mouse line. Immunofluorescence deconvolution microscopy will be used to assess overall sarcomere structure and changes in thin filament lengths in these KO mice. Contractile force of individual skeletal and cardiac myocytes will be measured. We will attempt to prevent skeletal and cardiac defects in these mice by introducing Lmod3 via adeno-associated virus. Aim 2 will determine the effect of a nemaline myopathy-linked mutation on thin filament lengths and actin dynamics. Mutated Lmod and Tmod proteins will be expressed in both skeletal and cardiac myocytes via adenovirus. Thin filaments will be visualized and measured using immunofluorescence microscopy, while fluorescence recovery after photobleaching will test mutated Lmod and Tmod's ability to assemble to the pointed ends. Aim 3 is to determine how a nemaline myopathy-linked mutation affects structure and function of Lmod/Tmod. Circular dichroism will be used to investigate the ability of mutated Lmod and Tmod to fold properly, and nuclear magnetic resonance will be used to determine how structural alterations could potentially affect mutated Tmod and Lmod's binding interfaces with other proteins, such as actin and tropomyosin. We will assess functional changes in mutated Lmod and Tmod by performing pyrene-actin polymerization assays and co-sedimentation assays. The long-term goal of this multidisciplinary project, that spans from single molecule to whole animal studies, is to determine how perturbation of actin-thin filament lengths leads to muscle disease. This is significant because actin is the most abundant protein in most cell types and is involved in numerous essential cellular processes. The results obtained in this project will allow us to decipher the connection between thin filament lengths and muscle function, and in vivo mechanistic information on how a single mutation in Lmod3 leads to human myopathy.