Examining the Actin Regulatory Functions of Pointed End Binding Proteins in Striated Muscle

Abstract

Striated muscle cells are composed of repeating contractile units known as sarcomeres. Contraction of the muscle occurs when myosin (thick) filaments and actin (thin) filaments overlap and bind to one another. The lengths of thin and thick filaments are critical for efficient contractile activity. However, the mechanism underlying thin filament assembly and regulation is largely unknown. Tropomodulins (Tmods) and leiomodins (Lmods) are actin-binding proteins within the same family. These proteins have been shown to play an important role in regulating the lengths of thin filaments by modulating actin polymerization at their pointed ends (near the center of the sarcomere). Although having overall high sequence homology, Tmod and Lmod notably possess opposite functions in which Tmod restricts while Lmod elongates thin filament lengths. Alterations in the lengths of thin filaments as well as mutations in Tmod and Lmod have been linked to the development of both cardiac and skeletal myopathies. Thus, it is necessary to study the cellular and molecular functions of Tmod and Lmod in order to better understand the mechanisms underlying muscle diseases. In this dissertation, Tmod and Lmod’s actin regulatory functions were studied by introducing a clinically relevant point mutation in the striated muscle isoforms of these proteins. This mutation was previously identified in the Lmod3 isoform in patients with nemaline myopathy, a debilitating genetic muscle disorder characterized by extreme skeletal muscle weakness and hypotonia. This heterozygous missense mutation results in an amino acid change from a glycine to an arginine (G326R), which we have defined as the G-to-R mutation. G326 resides in Lmod3’s leucine-rich repeat (LRR) domain which is predicted to bind actin. Interestingly, this amino acid is also conserved in Tmod1 and Lmod2 G268 and G291, respectively. In order to determine the physiological function of this residue and LRR domain, we introduced this G-to-R mutation in these proteins. Remarkably, we found that this single mutation disrupts both Tmod and Lmod’s ability to effectively regulate filament lengths in cells. We also discovered that Tmod’s ability to cap pointed ends of actin filaments and Lmod’s ability to nucleate actin polymerization are impaired with this mutation. Additionally, we revealed that expression of the mutation leads to disorder in the structure of the homologous LRR domains of Tmod and Lmod and, consequently, weakens their interaction with actin by altering their binding interface. By utilizing an Lmod3 knockout mouse model, we found that this mutation renders Lmod3 nonfunctional in vivo. Specifically, while exogenous Lmod3 expression is able to prevent the development of several nemaline myopathy disease phenotypes that are exhibited in Lmod3 knockout (KO) mice, expression of exogenous mutant Lmod3 expression was not. In summary, we demonstrated how exceptionally damaging the nemaline myopathy-linked (G-to-R) mutation is to the structures and respective actin-regulatory functions of Tmod and Lmod as well as reveal that the homologous LRR domain is crucial for maintaining these functions. This investigation reveals that at the molecular, cellular, and in vivo levels one single amino acid is essential for Tmod’s ability to shorten and Lmod’s ability to elongate thin filament lengths. Moreover, weakening of Lmod3’s interaction with actin could likely provide the mechanism underlying the development of certain forms of nemaline myopathy.