The long term objective of this research is to identify the molecular components and mechanisms that regulate contractile protein interactions during thin filament assembly: properties required for the proper functioning of cardiac muscle cells. The goal of this proposal is to define the in vivo function(s) of tropomodulin, the only known actin filament pointed capping protein, and its specific molecular interactions in the regulation of thin filament assembly, length and organization. Base on recent studies, the applicant hypothesizes that the tropomyosin-binding, actin filament capping, and nebulin-binding domains of tropomodulin serve different functions and are responsible for different physiological properties in vivo.
The Aims are: 1) To examine tropomyosin-tropomodulin interactions and to identify other molecular interactions required for actin filament capping, by microinjecting Fab fragments of defined, function-blocking monoclonal antibodies into embryonic chick cardiac myocytes. In addition, truncated tropomodulins that can act as dominant negative inhibitors will be overexpressed. Thin filament length and organization will be examined by immunofluorescence and electron microscopy, and contractile function by video microscopy. 2) To determine the role of tropomodulin in myofibril assembly by inappropriately increasing or reducing tropomodulin protein levels by using antisense techniques or by microinjection of antibodies to crosslink and sequester tropomodulin. 3) To identify the domains of tropomodulin and of tropomyosin isoforms that are required for the assembly of tropomodulin onto actin filament pointed ends using a permeabilized cell model and microinjection of tracer levels of tropomodulin fragments. 4) To identify a putative cardiac nebulin-like molecule: characterize and determine the functional significance of its interaction with tropomodulin.
| Conover, Gloria M; Gregorio, Carol C (2011) The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments. J Cell Sci 124:3464-76 |
| Pappas, Christopher T; Krieg, Paul A; Gregorio, Carol C (2010) Nebulin regulates actin filament lengths by a stabilization mechanism. J Cell Biol 189:859-70 |
| Conover, Gloria M; Henderson, Syerra N; Gregorio, Carol C (2009) A myopathy-linked desmin mutation perturbs striated muscle actin filament architecture. Mol Biol Cell 20:834-45 |
