The aim is to determine how muscle contraction is controlled by studying the regulation of muscle thin filaments. The control of Drosophila flight muscle thin filaments by calcium and stretch is being investigated. The biochemical properties of the isolated components of the regulatory complex, troponin, are being studied. The function of the different domains in the sequence of the molecules is found by expressing partial sequences in E. coli; calcium binding and interaction with other thin filament components is then investigated.The function of isoforms of regulatory proteins and of domains in the proteins is studied by the techniques of molecular genetics. Mutations in the regulatory protein genes will reveal the function of particular regions of the molecules; anti-sense constructs will be used to reduce or abolish expression of gene products. The function of isolated thin filaments from these mutants will be studied as well as the mechanical properties of the muscles and flight capability of the fly.The structure of thin filaments and the position of troponin is determined by low dose cryo-electron microscopy and the shape of the isolated troponin molecule will be found by shadowing techniques. The function of thin filaments is studied by using optical tweezers. These methods will be used with thin filaments in which troponin components are altered or omitted.
Thin filaments have been isolated from a Drosophila mutant which lacks thick filaments and changes in the structure of the filaments on calcium binding has been determined by electron microscopy. The presence of troponin (TnI)sequence in troponinH (TnH) has been confirmed immunologically and calcium binding and phosphorylation of troponin T (TnT) have been demonstrated. TnH has been expressed in E coli and binding to other proteins studied. Suppressor mutants have been characterised which rescue mutants in the TnI gene. One is within the TnI gene itself, one is in the myosin gene and another is probably in a tropomyosin gene. Suppressors reveal interactions between TnI and other proteins. Two genes related to TnI have been sequenced and located in the nervous ssytem and gonads. A chromosome inversion within a tropomyosin gene has been found in a mutant and the effect on muscle structure will be studied.
Funding SchemeCSC - Cost-sharing contracts
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