Journal of Molecular Biology
Alternative Relay and Converter Domains Tune Native Muscle Myosin Isoform Function in Drosophila
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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2020, Seminars in Cell and Developmental BiologyCitation Excerpt :The 11a domain has the fastest kinetics and highest power generation of all five converter domains, while the embryonic 11d converter has reduced ATPase activity but supports increased actin sliding velocity [141]. Interestingly, rescue of Mhc10 mutant IFM with an Mhc isoform containing both the embryonic converter (11d) and relay (9c) domains, which together regulate ATPase activity and lever-arm positioning of the myosin head, results in abnormal myofibril assembly [142,143]. Moreover, expression of the slow muscle hinge (15b) instead of the IFM variant (15a) increases myosin rod and sarcomere length, but decreases wing-beat frequency and flight performance [144].
X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties
2020, Journal of Molecular BiologyCitation Excerpt :Both of the portions encoded for in exon 11 that mediate these contacts are highly conserved between EMB and IFI (Supplementary Fig. 1). However, altered properties of the converter that arise as a consequence of differences in EMB exon 11c and IFI exon 11e could influence myosin action at a distance through a network communicating the nucleotide binding state to the lever arm through exons 9, 11, and the ELC [67]. Mutation of Arg756 from the converter to Glu was previously shown to correlate with a 60% decrease in ATPase rates and 35% decrease in actin sliding motility as well as loss of flight ability without affecting actin binding affinity.
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2019, International Journal of Biochemistry and Cell BiologyCitation Excerpt :The slow myosin hinge region encoded by MHC exon 15b is stiffer than the fast hinge region encoded by MHC exon 15a, and swapping exon 15a and 15b impairs flight ability, increases sarcomere length and disrupts sarcomere structure (Suggs et al., 2007). Differences in force generation, ATPase activity and rate-limiting cross-bridge steps between embryonic and flight muscle MHC isoforms can be attributed to the relay (exon 9) and converter (exon 11) domains, which also differentially impact myofibril ultrastructure (Kronert et al., 2012; Yang et al., 2010, 2008). These studies illustrate that from flies to mammals, different MHC isoforms confer distinct biophysical properties on the sarcomere and differentially affect myofibril development.
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2015, Journal of Biological ChemistryCitation Excerpt :A remarkable aspect of our analysis is that ultrastructural defects in sarcomere assembly can arise from mutations in the relay or converter domains. This is consistent with our previous studies on mutants and isoform domain switches (19, 35, 36), and suggests that a functional relay-converter interface permits necessary interactions required for myofibrillogenesis. Structural defects may be exacerbated by altered actin-myosin interactions during attempted muscle use.
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2014, Experimental Cell ResearchCitation Excerpt :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]. Thus, alterations in Mhc splicing in IFMs would affect myofibril assembly and stability and also change the fine-tuning of the myosin ATPase and actin filament motility.