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

项目总结

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.