Striated muscles represent the pinnacle of cytoskeletal organization among cells, with highly ordered arrays of actin and myosin filaments in tightly arranged sarcomeres, all poised to respond to electrochemical signals with cell-wide coordinated contraction. Genetic defects in sarcomeric proteins result in debilitating myopathies of heart and skeletal muscle. Understanding how these complex cytoskeletal structures normally assemble will be vital to therapeutic resolution of these diseases. Formins are ubiquitous actin-organizing proteins that play a critical but poorly understood role in directing sarcomere formation. In mice, the formin FHOD3 is required for mature sarcomere formation in heart muscle cells required for embryonic development, and a naturally occurring variant of the FHOD3 gene in humans is tied to an increased incidence of hypertrophic cardiomyopathy, the most common cause of sudden cardiac-related deaths in young adults. A critical barrier to understanding how FHOD3 functions is that loss of the formin likely has immediate (short- term) as well as indirect (long-term) consequences that cannot currently be resolved in mammalian systems. To understand how FHOD3 and other formins contribute to sarcomere organization, we are employing the powerful genetic model system Caenorhabditis elegans. Sarcomere assembly in this roundworm is also promoted by an FHOD3-related formin FHOD-1, and a second formin CYK-1. Using this model, we will address three fundamental questions: 1) Do formins direct the assembly of the actin component of sarcomeres? Using worms with conditional formin mutations, we will test whether an acute loss of formin activity blocks actin filament assembly in developing sarcomeres. 2) Is formin-dependent actin filament nucleation needed for sarcomere assembly? We will design mutations that disrupt the in vitro nucleating activites of FHOD-1 and CYK-1, and determine whether these mutations block the ability of these formins to promote sarcomere formation in vivo. 3) How does formin loss affect non-actin components of sarcomeres? Muscle myosin heavy chain IIA expression depends on FHOD-1 activity. We will dissect the mechanism of this regulation as an archetype of how formin activity can control the assembly of non-actin sarcomeric components in vivo. With these experiments, we will reveal the mechanisms by which these highly conserved proteins direct the core process of sarcomere formation in striated muscle, which is key to understanding and treating diseases of these complicated and vital cells.
Muscle function depends on small, contractile units within muscle cells called sarcomeres. Defects in proteins that make up the sarcomeres result in debilitating myopathies (diseases) of voluntary and heart muscle, and discovering how sarcomeres assemble is key to treating such myopathies. In this proposal, we will determine how essential protein drivers of sarcomere formation called formins direct sarcomere assembly in muscle.