Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7.

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell-cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

Multinucleated smooth muscles and mononucleated as well as multinucleated striated muscles develop during establishment of the male reproductive organs of Drosophila melanogaster.

The adult musculature in D. melanogaster forms during metamorphosis. Much is known about the flight and leg musculature, but not about the muscles surrounding the male reproductive tract. The inner genitalia of males consist of the testes, which emerge from the gonads; the remaining genital organs, i.e., paragonia (or accessory glands), ejaculatory duct, sperm pump, and seminal vesicles, develop out of the genital imaginal disc. We analyzed the myoblasts forming the muscle layers of these organs. In myoblasts derived from the genital imaginal disc, the regulatory region of the transcription factor DMef2 is active. DMef2 is also needed for specification and differentiation of embryonic and adult myoblasts. We could discriminate three different muscle types: (i) multinucleated muscles that resemble vertebrate smooth muscles surround the testes, (ii) multinucleated muscles that resemble striated muscles comprises seminal vesicles and the sperm pump, and (iii) mononucleated striated musculature encloses the paragonia and ejaculatory duct. Members of the immunoglobulin superfamily involved in embryonic myogenesis, Dumbfounded (Duf) and Sticks and Stones (Sns), were also expressed in the genital imaginal disc, in the muscle sheath of the testes during muscle differentiation and in the secretory secondary cells, which are part of the binucleated epithelia enclosing the paragonia.

Drosophila Swiprosin-1/EFHD2 accumulates at the prefusion complex stage during Drosophila myoblast fusion.

In the Drosophila embryo, transient cell adhesion during myoblast fusion is known to lead to the formation of fusion-restricted myogenic-adhesive structures (FuRMASs). Here, we report that within these FuRMASs, a Drosophila homologue of human and mouse swiprosins (EF-hand-domain-containing proteins) is expressed, which we named Drosophila Swiprosin-1 (Drosophila Swip-1). Drosophila Swip-1 is highly conserved and is closely related to the calcium-binding proteins swiprosin-1 and swiprosin-2 that have a role in the immune system in humans and mice. Our study shows that Drosophila Swip-1 is also expressed in corresponding cells of the Drosophila immune system. During myoblast fusion, Drosophila Swip-1 accumulates transiently in the foci of fusion-competent myoblasts (FCMs). Both the EF-hand and the coiled-coil domain of Drosophila Swip-1 are required to localise the protein to these foci. The formation of Drosophila Swip-1 foci requires successful cell adhesion between FCMs and founder cells (FCs) or growing myotubes. Moreover, Drosophila Swip-1 foci were found to increase in number in sing(22) mutants, which arrest myoblast fusion after prefusion complex formation. By contrast, Drosophila Swip-1 foci are not significantly enriched in blow(2) and kette(J4-48) mutants, which stop myogenesis beyond the prefusion complex stage but before plasma membrane merging. Therefore, we hypothesise that Drosophila Swip-1 participates in the breakdown of the prefusion complex during the progression of myoblast fusion.