Alternative Relay and Converter Domains Tune Native Muscle Myosin Isoform Function in Drosophila

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Abstract

Myosin isoforms help define muscle-specific contractile and structural properties. Alternative splicing of myosin heavy chain gene transcripts in Drosophila melanogaster yields muscle-specific isoforms and highlights alternative domains that fine-tune myosin function. To gain insight into how native myosin is tuned, we expressed three embryonic myosin isoforms in indirect flight muscles lacking endogenous myosin. These isoforms differ in their relay and/or converter domains. We analyzed isoform-specific ATPase activities, in vitro actin motility and myofibril structure/stability. We find that dorsal acute body wall muscle myosin (EMB-9c11d) shows a significant increase in MgATPase Vmax and actin sliding velocity, as well as abnormal myofibril assembly compared to cardioblast myosin (EMB-11d). These properties differ as a result of alternative exon-9-encoded relay domains that are hypothesized to communicate signals among the ATP-binding pocket, actin-binding site and the converter domain. Further, EMB-11d shows significantly reduced levels of basal Ca- and MgATPase as well as MgATPase Vmax compared to embryonic body wall muscle isoform (EMB) (expressed in a multitude of body wall muscles). EMB-11d also induces increased actin sliding velocity and stabilizes myofibril structure compared to EMB. These differences arise from exon-11-encoded alternative converter domains that are proposed to reposition the lever arm during the power and recovery strokes. We conclude that relay and converter domains of native myosin isoforms fine-tune ATPase activity, actin motility and muscle ultrastructure. This verifies and extends previous studies with chimeric molecules and indicates that interactions of the relay and converter during the contractile cycle are key to myosin-isoform-specific kinetic and mechanical functions.

Graphical Abstract

Research Highlights

► Myosin isoforms help define muscle-specific contractile and structural properties. ► We expressed three native embryonic myosin isoforms in Drosophila muscles. ► Native isoforms differ in their relay and/or converter domains. ► Isoforms yield unique ATPases, in vitro actin motilities and myofibril structures. ► Alternative relay and converter domains fine-tune myosin function.

Section snippets

Background

While vertebrates have muscle myosin heavy chain (MHC) gene families, Drosophila melanogaster alternatively splices transcripts from its single muscle Mhc gene to produce multiple MHC isoforms that are expressed in a muscle-specific fashion.1, 2, 3, 4, 5 The Drosophila system is advantageous in that it highlights specific alternatively encoded protein domains that are responsible for myosin functional diversity. Mhc contains two alternative versions of exon 3, four of exon 7, three of exon 9,

P element transformation and generation of transgenic lines

The EMB-11d and EMB-9c11d transgenes contain the 5′ and 3′ ends of Mhc along with a cDNA insert encoding alternative embryonic MHC isoforms. These were constructed as detailed in Materials and Methods. Transgenic lines that express each of the embryonic isoforms were produced by embryo injection. To analyze transgenic myosin in the absence of endogenous MHC, we crossed each first, third or fourth chromosome transgenic line into the Mhc10 background, which is null for MHC in IFM and jump muscle.

Discussion

In this paper, we compared the biochemical, biophysical and in vivo assembly/stability properties of three naturally occurring Drosophila MHC isoforms: EMB-9c11d is normally expressed in the dorsal acute embryonic body wall muscles, and EMB-11d is expressed in embryonic cardioblasts (as well as in dorsal acute and ventral oblique body wall muscles), whereas EMB is expressed in eight different types of body wall muscles.5 These three isoforms differ only in their alternative relay and/or

Protein structure analysis

The scallop muscle myosin II crystal structure in the pre-power stroke state (Protein Data Bank ID 1qvi) and post-power stroke state (Protein Data Bank ID 1kk8) were used as templates for the Drosophila embryonic myosin S-1 amino acid sequence. The Swiss-Model homology modeling server† was used to produce the homology coordinates, and PyMOL‡ was employed to view the output produced by the homology server. We also used this approach to view the

Acknowledgements

Funds to support this research were provided by the National Institutes of Health grant R01 GM32443 to S.I.B. A National Science Foundation equipment grant (0308029) to Dr. Steven Barlow of the San Diego State University Electron Microscope Facility supported the purchase of the electron microscope. We appreciate the efforts of Jennifer Suggs in performing the crosses for the crawling assays. We thank Dr. Douglas Swank (Rensselaer Polytechnic Institute) for helpful comments on the manuscript.

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