Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice

Abstract

SummaryDuring muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by nontranslation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

Sallimus and the Dynamics of Sarcomere Assembly in Drosophila Flight Muscles

“…Therefore, beyond simply connecting the Z-disc to the thick filaments, sallimus is a direct and necessary structural component of the Z-disc. Supporting our previous work [12], we also provide evidence that thick filament length and symmetry specification takes place within the growing sarcomere and that the Z-disc is required for this. Knockdown of the M-line protein obscurin in IFM, which led to mis-formed M-lines, resulted in asymmetric thick filaments [9], showing that the M-line is also needed for assembly of symmetrical thick filaments.…”

“…All this supports the premyofibril model of sarcomere assembly [1][2][3] for Drosophila IFM, in which all sarcomere components assemble together, whilst arguing against the notion of thick filaments forming independently before their incorporation into the myofibril. However, in IFM, each sarcomere assembles independently, without lateral fusion of nascent myofibrils [4,5,12] in contrast to the premyofibril model for vertebrate myofibril assembly [15,16]. On the other hand, we show that the Z-disc is not required for thin/ thick filament lattice formation.…”

Roles of the troponin isoforms during indirect flight muscle development in Drosophila

“…Most of the structural proteins of the IFMs are homologous to their vertebrate counterparts, performing similar function during muscle contraction (Vigoreaux 2006). A majority of these proteins undergo isoform switch during later stages of development to meet the physiological demands of the adult flight (Marden 2006;Orfanos and Sparrow 2013). The IFMs are dispensable for survival under laboratory conditions, providing an effective genetic system to study the developmental and functional importance of different isoforms (Nongthomba et al 2004;Vigoreaux 2006).…”

“…There is no reported mutation for either TnC1 or TnC4, though biochemical studies suggest that TnC1 is required for isometric contraction and TnC4 for stretch activation (Linari et al 2004;Krzic et al 2010;Bullard and Pastore 2011). Most of the Isoforms switch during later stages of IFM development, around 65-75 hours after puparium formation (hAPF) (Nongthomba et al 2004;Orfanos and Sparrow 2013). Developmental and functional consequences of reduction in the expression of specific troponin isoforms during the isoform-switching stage have not been addressed before.…”

Transcriptional regulation and alternative splicing cooperate in muscle fiber-type specification in flies and mammals

“…Alternative splicing generates the wide diversity of Mhc protein isoforms expressed in Drosophila muscle fibers. IFMs express a different complement of alternatively spliced exons than larval Mhc isoforms, resulting in Mhc proteins with distinct physiological properties [56,76]. In particular, the relay domains encoded by exon 9 variants result in variations in MgATPase activity and actin sliding velocity and affect myofibril assembly and stability, while variants in the converter domain encoded by exon 11 affect CaATPase, MgATPase, and actin sliding velocity [38].…”