However, the functional regions of tropomodulin are not organized in a simple modular fashion along the amino acid sequence, because previous studies showed that residues in the N-terminal half are also important for binding to tropomyosin. We recently began crystallization trials to obtain an x-ray crystal structure for tropomodulin, and we are developing methods to obtain electron microscopic images of tropomodulin bound to the pointed ends of thin filaments. To investigate the molecular basis for tropomodulin function in striated muscle in vivo, we are using antibody inhibition and dominant negative or molecular genetic strategies to alter the levels of tropomodulin and selectively interfere with its interactions with actin, tropomyosin, or other components. Effects on the length and organization of actin filaments are evaluated by using immunofluorescence staining, confocal microscopy, and thin-section electron microscopy. Effects on the monomer-polymer dynamics of the filaments are evaluated by monitoring incorporation of microinjected, fluorescently labeled actin into the actin filaments of living muscle cells.
Striated muscle is an extreme example of cytoskeletal architecture.
Nevertheless, we anticipate that the components and mechanisms that control the activity of tropomodulin and the dynamics of actin filaments to produce filaments with uniform lengths in striated muscle will be broadly applicable to red blood cells and other cell types. Recent data from our laboratory and other laboratories indicate that tropomodulins are a rapidly growing family of related proteins expressed in many vertebrate tissues and in flies and worms. We are using biochemical and molecular approaches to investigate the functional activities of tropomodulin isoforms. We are examining capping of actin filaments and binding of tropomyosin and are determining novel interaction partners that might regulate the activities of tropomodulin isoforms differentially in various tissues or during development. A particular focus is the role of tropomodulin isoform switching and function in fiber cell differentiation and morphogenesis in the chick eye lens.