The applicant proposes to increase our understanding of the regulatory circuitry that controls the proliferation, fusion and differentiation of skeletal myoblasts to form muscle fibers during development and aging. In an effort to extend the understanding of the regulation of myoblast fusion, an early step in muscle tissue formation and regeneration the applicant proposes to determine rigorously the effects of certain cell surface molecules, substrates, and growth factors on the proliferation and differentiation of myoblasts in tissue culture using novel assays. The applicant also proposes to study the heterodimerization of members of the helix-loop helix family of myogenic regulators in live proliferating and differentiating muscle cells. The test system will involve chimeric proteins composed of these regulators and complementing DATE - galactosidase (DATE -gal) deletion mutants, and will permit analysis of heterodimerization and nuclear translocation in the intact cell. This approach will serve as a powerful adjunct to the in vitro methods routinely used for studying dimerization by examining these interactions in a biologically relevant context, the intact cell. The applicant will assess in mice which of the expressed myogenic activators and inhibitors actually interact to form heterodimers during development. A few key regulators will be selected for study in vivo using a knock in of a construct containing a myogenic regulator-DATE -gal chimera, and heterodimerization will be assayed by DATA -gal complementation. This will determine, for example, whether and when twist and Myf5 which are co- expressed in the developing mouse embryo, heterodimerize to prevent myogenesis. The experiments outlined in this proposal are possible by the recent adaptation of DATE -gal complementation in mammalian cells. The fusion of two myoblasts expressing strongly complementing deletion mutant peptides of DATE -gal results in DATE - gal enzyme activity that provides a rapid, reproducible, and quantitative measure of cell fusion. Similarly, DATE -gal activity resulting from the interaction of two chimeric proteins containing weakly complementing DATE -gal peptides measures the extent of interaction of the non-DATE -gal portions of the chimeras in an intact cell. These assays provide critical new tools for use in the proposed studies that will contribute to our understanding of the regulatory mechanisms underlying muscle development and aging.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD018179-17
Application #
6138752
Study Section
Molecular Cytology Study Section (CTY)
Program Officer
Javois, Lorette Claire
Project Start
1983-07-01
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
17
Fiscal Year
2000
Total Cost
$297,263
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Wang, Heng; Lööf, Sara; Borg, Paula et al. (2015) Turning terminally differentiated skeletal muscle cells into regenerative progenitors. Nat Commun 6:7916
Palermo, Adam; Doyonnas, Regis; Bhutani, Nidhi et al. (2009) Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional. FASEB J 23:1431-40
Pomerantz, Jason H; Mukherjee, Semanti; Palermo, Adam T et al. (2009) Reprogramming to a muscle fate by fusion recapitulates differentiation. J Cell Sci 122:1045-53
von Degenfeld, Georges; Wehrman, Tom S; Blau, Helen M (2009) Imaging beta-galactosidase activity in vivo using sequential reporter-enzyme luminescence. Methods Mol Biol 574:249-59
Pajcini, Kostandin V; Pomerantz, Jason H; Alkan, Ozan et al. (2008) Myoblasts and macrophages share molecular components that contribute to cell-cell fusion. J Cell Biol 180:1005-19
Johansson, Clas B; Youssef, Sawsan; Koleckar, Kassie et al. (2008) Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation. Nat Cell Biol 10:575-83
Keshet, Gilmor I; Tolwani, Ravi J; Trejo, Angelica et al. (2007) Increased host neuronal survival and motor function in BMT Parkinsonian mice: involvement of immunosuppression. J Comp Neurol 504:690-701
von Degenfeld, Georges; Wehrman, Tom S; Hammer, Mark M et al. (2007) A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. FASEB J 21:3819-26
Hammer, Mark M; Wehrman, Tom S; Blau, Helen M (2007) A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. FASEB J 21:3827-34
Kutschka, Ingo; Chen, Ian Y; Kofidis, Theo et al. (2007) In vivo optical bioluminescence imaging of collagen-supported cardiac cell grafts. J Heart Lung Transplant 26:273-80

Showing the most recent 10 out of 88 publications