Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.
Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.
Swank
D M
DM
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182-4614, USA.
Wells
L
L
Kronert
W A
WA
Morrill
G E
GE
Bernstein
S I
SI
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D009210:Myofibrils / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012518:Sarcomeres / Q000378:metabolism*; D013329:Structure-Activity Relationship; D019076:Transgenes
D018995:Myosin Heavy Chains