Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta

Abstract

The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

The effect of muscle ultrastructure on the force, displacement and work capacity of skeletal muscle

“…As a result, titin has been suggested to affect muscle force, work and power particularly when the muscle has previously been actively stretched or shortened [15][16][17][18][19]25,91]. The location and size of titin-like proteins appear to vary considerably across invertebrate muscles [83][84][85][86][87][88][89][90], and unlike in vertebrates, very little is known about their active function with the exception of the highly specialized molluscan twitchin [92]. The role of titin in active muscle, and the variation in titin-like proteins likely has implications for our predictions of force and work.…”

“…However, we also lack information about the role of these features of muscle in contraction, a knowledge of the variation in these features across muscles, empirically determined force-length relationships for known actin and myosin filament lengths, and an understanding of the mechanisms responsible for the ascending limb of the force-length relationship. We systematically exclude titin, and the various isoforms of the array of titin-like proteins observed in invertebrates [83][84][85][86][87][88][89][90], from this model. In vertebrates, this large viscoelastic protein [15,25] spans entire half sarcomeres from the Z-disc to M-line [20] and increases stiffness upon muscle activation both by binding to actin [26], and potentially by being wound onto actin filaments by cross-bridge cycling [25,26].…”