In vitro motility of native thin filaments from Drosophila indirect flight muscles reveals that the held-up 2 TnI mutation affects calcium activation

A procedure for the isolation of regulated native thin filaments from the indirect flight muscles (IFM) of Drosophila melanogaster is described. These are the first striated invertebrate thin filaments to show Ca-regulated in vitro motility. Regulated native thin filaments from wild type and a troponin I mutant, held-up-2, were compared by in vitro motility assays that showed that the mutant troponin I caused activation of motility at pCa values higher than wild type. The held-up2 mutation, in the sole troponin I gene (wupA) in the Drosophila genome, is known to cause hypercontraction of the IFM and other muscles in vivo leading to their eventual destruction. The mutation causes substitution of alanine by valine at a homologous and completely conserved troponin I residue (A25) in the vertebrate skeletal muscle TnI isoform. The effects of the held-up 2 mutation on calcium activation of thin filament in vitro motility are discussed with respect to its effects on hypercontraction and dysfunction. Previous electron microscopy and 3-dimensional reconstruction studies showed that the tropomyosin of held-up 2 thin filaments occupies positions associated with the so-called 'closed' state, but independently of calcium concentration. This is discussed with respect to calcium dependent regulation of held-up-2 thin filaments in in vitro motility.