Effect Of Down-Regulation of a Stretch-Activated TnC Isoform on Flight of Drosophila

Abstract

Both Drosophila and Lethocerus have indirect flight muscle (IFM) that is activated by sinusoidal length changes at constant [Ca2+]. IFM has two TnC isoforms. F1 binds a single Ca2+ in the C-lobe and is needed for the periodic stretch-activation of fibres to produce oscillatory work. F2 binds Ca2+ in both N- and C-lobes and is needed for producing Ca2+-dependent isometric tension. We have obtained flies (from VDRC, Vienna) in which F1 is down-regulated by RNAi. Male flies of the F1 RNAi line were crossed with virgin female flies having the Dmef2 driver, which is expressed in all muscles, or a UH3 driver, which is expressed only in IFM. Crosses were maintained at 25oC and 29oC to get different levels of RNAi expression. The proportion of flies unable to fly was: wt 0%; Dmef2 87% at 25oC, 90% at 29oC; UH3 70% at 25oC, 100% at 29oC. There was no difference in time of development or viability of the different lines. Confocal microscopy of Dmef2 and UH3 flies showed myofibrils of both lines were narrower than wt; sarcomere length was normal, but Z-disc and M-line were not straight. Electron microscopy showed that sarcomere structure was disrupted more than expected. Troponin was regularly spaced at 38 nm along thin filaments, but thick and thin filaments were misaligned and Z-and M-lines shifted. Blots of IFM with anti-F1 and F2 showed F1was absent in Dmef2 flies, and greatly reduced in UH3 flies; F2 content of IFM was the same as wt. Therefore, F1 is essential for maintaining normal sarcomere structure of IFM, as well as for stretch-activation. Evidence for cross-linking between troponin components and thick filaments of Lethocerus IFM will be presented. Lack of F1 may affect these links.