Our long-term goals are to understand how sarcomeric proteins are assembled into myofibrils, and, how when mutated, truncated or deleted, these sarcomeric proteins result in myopathies. The experiments focus on Z-band proteins that have an essential role in the formation and maintenance of myofibrils. Our first hypothesis is that as z-bodies develop into Z- bands, the increasing structural organization is accompanied by changes in the dynamics and binding interactions of the proteins resulting in a structure capable of supporting contractions. The major strategy of our experimental approaches in this proposal is to analyze the formation of the Z-bands of myofibrils inside living skeletal muscle cells via probes encoding fluorescent chimeric GFP-muscle proteins. The first specific aim is to investigate the assembly, the dynamics and proximities of several proteins in the z-bodies and Z-bands in skeletal myocytes in living zebrafish by using various microscopical approaches. Confocal and deconvolution microscopy will be used to follow the assembly of GFP-sarcomeric proteins during myofibrillogenesis in living zebrafish. FRAP (Fluorescence Recovery After Photobleaching) experiments will measure the changing dynamics of key proteins in the z-bodies and Z-bands during myofibrillogenesis. Steady-state FRET (Fluorescence Resonance Energy Transfer) efficiencies of the proteins and their binding partners co-expressed in living skeletal muscle cells will be analyzed to produce a detailed picture of protein interactions during Z-band formation. There are a number of myopathies in which Z- band proteins are mutated. Our second hypothesis is that mutations of actin, myotilin, ZASP/cypher and telethonin, or deletions of telethonin will lead to altered dynamics and binding properties of proteins in the Z-band, and other parts of the I-bands, changing the stability of the myofibrils. The second specific aim is to analyze, on the single cell level, the effects of several mutations of these four Z-band proteins known to be involved in myopathies. FRAP, FRET and biochemical analyses will be used to determine if the mutations affect the dynamics and interactions of the mutated proteins and their binding partners (alpha-actinin, FATZ, myotilin) in the Z-bands, and in the thin filaments in the I-bands (tropomyosin), and thus reveal the molecular bases for the muscle disease in living muscle cells. Our experiments should yield novel insights into myofibril assembly, maintenance, and myopathies.

Public Health Relevance

Myofibril assembly exemplifies cellular engineering of exacting precision that produces an array of proteins dedicated to regulated force production in skeletal muscles. Although the component proteins are arranged in a precise structural framework, they are dynamic and capable of exchanging between the myofibril and a cellular pool of proteins. We think that mutated proteins associated with skeletal muscle diseases have altered dynamic and binding properties that lead to an instability of the myofibrils. Myofibrils containing these mutated proteins cannot support the force production, and thus the myofibrils become unstable and fall apart.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR057063-05
Application #
8464634
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2009-07-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$318,942
Indirect Cost
$115,794
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
White, Jennifer; Barro, Marietta V; Makarenkova, Helen P et al. (2014) Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes. Anat Rec (Hoboken) 297:1571-84
Sanger, Jean M; Sanger, Joseph W (2014) Recent advances in muscle research. Anat Rec (Hoboken) 297:1539-42
Dube, Dipak K; Wang, Jushuo; Pellenz, Christopher et al. (2014) Expression of myotilin during chicken development. Anat Rec (Hoboken) 297:1596-603
Wang, Jushuo; Fan, Yingli; Dube, Dipak K et al. (2014) Jasplakinolide reduces actin and tropomyosin dynamics during myofibrillogenesis. Cytoskeleton (Hoboken) 71:513-29
Wang, Jushuo; Dube, Dipak K; Mittal, Balraj et al. (2011) Myotilin dynamics in cardiac and skeletal muscle cells. Cytoskeleton (Hoboken) 68:661-70