The Influence of Troponin C Isoforms on Drosophils Stretch Activation and Power Generation

Abstract

Drosophila indirect flight muscle (IFM) is one of the most rapidly contracting muscle types known. To efficiently generate power for flight at such high speeds, insects have evolved stretch activation and shortening deactivation. In IFM of some insects, in addition to a typical calcium binding troponin C, TnC1, there is a unique, additional isoform, TnC4. TnC1 has 2 calcium binding sites, a high-affinity structural site and a lower-affinity regulatory site. TnC4 has only the high-affinity structural binding site. It is hypothesized to respond to stretch rather than Ca2+ concentration to further activate the thin filament. We are investigating TnC4 in Drosophila via two different routes. The first is to chemically remove TnC from IFM using either o-vanadate or trifluoperazine which eliminates power generation and stretch activation. Bacterially-expressed TnC1 and TnC4 are reincorporated into the fiber in physiological proportions, which restores power-generating ability and stretch activation to the IFM. We are currently optimizing the extraction and reintroduction protocol to determine the effects on stretch activation of varying the ratio of TnC1 and TnC4. The second method is to breed a TnC4 null using p-element mediated male homologous recombination. We are performing this by crossing flies containing identical p-elements in positions which bracket the TnC4 gene on chromosome 2R. When the p-elements are mobilized, some of the offspring will have the region between the p-elements excised. We are currently screening offspring of the final cross for null TnC4 alleles. These two methods of investigation into TnC4 function will allow modified TnC4 to either be introduced or expressed in IFM to help elucidate TnC4's structural mechanism.