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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR064760-01A1
Application #
8696239
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2014-04-01
Project End
2019-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
1
Fiscal Year
2014
Total Cost
$353,375
Indirect Cost
$133,375
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Hegsted, Anna; Wright, Forrest A; Votra, SarahBeth et al. (2016) INF2- and FHOD-related formins promote ovulation in the somatic gonad of C. elegans. Cytoskeleton (Hoboken) 73:712-728
Mangio, Richard S; Votra, SarahBeth; Pruyne, David (2015) The canonical eIF4E isoform of C. elegans regulates growth, embryogenesis, and germline sex-determination. Biol Open 4:843-51
Mi-Mi, Lei; Pruyne, David (2015) Loss of Sarcomere-associated Formins Disrupts Z-line Organization, but does not Prevent Thin Filament Assembly in Caenorhabditis elegans Muscle. J Cytol Histol 6: