A new tool for producing and investigating recombinant sarcomeric actin.

Abstract

Actin forms many cytostructural elements, including the backbone of striated muscle thin filaments. Our mechanistic understanding of most actin-dependent processes remains limited, partly due to difficulty with recombinant actin expression. While fully-functional actin cannot be produced in bacteria, it can be generated using baculovirus expression systems. Here, we created an additional tool for recombinant actin production, for hypothesis testing and for resolving the molecular properties and cellular effects of unique variants. Specifically, we engineered Drosophila that can express untagged actins exclusively in indirect flight muscles (IFMs). Act88F encodes sarcomeric IFM actin. Attempts to rescue IFM function (i.e. flight) in Act88F-nulls, by inserting transgenic Act88F (Act88FTG-WT) ectopically throughout the genome have failed, potentially due to ineffective cis/trans regulation of Act88FTG-WT expression. To circumvent these problems, we used CRISPR-editing and introduced an attP landing site directly into Act88F, to disrupt the endogenous gene and serve as a target for site-specific transgene insertion. attP-mediated disruption of Act88F yielded flightlessness and actin-free IFMs. Transformants expressing GFP or Act88FTG-WT, from the endogenous Act88F integration locus, demonstrated robust IFM-specific protein production and restored flight, respectively. Having established the expression system, we designed Drosophila mutants to test the roles of specific actin residues in tropomyosin/myosin-binding and nemaline myopathy pathogenesis. Both Act88FTG-R147Q and Act88F TG-F352Striggered IFM hypercontraction and impaired flight. Finally, we determined whether mammalian actin could be generated and observed high-level expression of human α-cardiac actin (ACTC1), which behaved indistinguishably from bovine ACTC1 in unregulated and regulated motility (IVM) assays. We are currently comparing IVM properties of IFM-purified vs. baculovirus/Sf12-purified ACTC1. Overall, our novel fly line permits transgenic actin expression in mature muscle fibers that are themselves amenable to structural and functional analyses, and serves as a cost-effective, perpetual source of ample protein for biophysical experimentation.