Exploring mechanisms of Troponin-T isoform switching and regulation of stoichiometry in the Troponin complex of Drosophila Indirect Flight Muscles. Aditi Madan, Divesh Thimmaiya, Prabodh Kumar, Upendra Nongthomba. MRDG, Indian Institute of Science, Bangalore, India.

   The indirect flight muscles (IFM) of Drosophila melanogaster have been used for decades to study muscle development and function. This is attributed to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Muscle contraction in striated muscles is regulated by the troponin (Tn) complex (Ca²⁺-binding troponin-C, inhibitory troponin-I & tropomyosin-binding troponin-T). These subunits are present in a 1:1:1 ratio on thin filaments. 2 isoforms of TnT are alternatively spliced in the Drosophila thorax - one containing alternative exon 10a (expressed in adult IFM & TDT); and one containing alternative exon 10b (expressed in pupae & newly eclosed flies). A splice mutant of TnT - up¹ - results in exclusion of the adult-specific TnT isoform (TnT-10a) and leads to defects in myofibrillogenesis. This study aims to understand functional differences between the 2 TnT isoforms, and elucidate probable mechanisms underlying the isoform switch. Transgenic UAS constructs have been used to rescue up¹, using muscle-specific Dmef2-GAL4 driver. Rescue has been analysed at 5 levels - physiological(flight tests), morphological(polarised light microscopy), myofibrillar(confocal microscopy), expression(RT-PCR) and protein(2D-GE). The homozygous rescued flies are flightless, but show rescue at structural and myofibrillar level. Rescue in the up¹/+ background shows recovery of flight ability as well as muscle structure. Control experiments were performed to test for the effect of over-expression of TnT in WT IFM. These experiments have shed light on the importance of stoichiometry amongst troponin subunits, as over-expression leads to flight defects. We have attempted to rescue up¹ using an alternative strategy - by knocking down a splicing factor reported to regulate the temporal switch from TnT-10b to TnT-10a in IFM. We have succeeded in partially rescuing flight ability and muscle architecture.