The role of PILMACs and of the genes bubbles and schwächling during muscle development in Drosophila melanogaster
Final Report Abstract
Cell-cell fusion is characteristic event during myogenesis – as for example also establishing the placenta or generatingosteoclasts for bone homoeostasis. We investigate myoblast fusion in Drosophila embryos as experimental model. Here, several fusion-competent myoblasts (FCMs) fuse with individual founder cells (FCs) that determine the identity of the each muscle. During our prework, we extensively characterised the spatial arrangement of fusion-relevant proteins. We found cell adhesion molecules forming a transient ring/belt connecting the FC/growing myotube with the FCM during fusion in the middle of which F-actin and its regulators accumulate in plugs/foci. We proposed that this belt of adhesion molecules surrounds the area of membrane breakdown. As vesicles accumulate at the membranes shortly before membrane fusion, we searched within this project for Calcium-binding proteins potentially involved in vesicle exocytosis. We identified Swiprosin-1/EFH2 (Swip-1) with two Calcium-binding EF hand domains, one coiled-coil domain and four predicated SH3-binding sites. Swiprosinis evolutionary highly conserved in mouse, human and Drosophila. Drosophila Swip-1 is mainly visible in FCMs during myoblast fusion and accumulates transiently in foci at the sites of contact between fusing myoblasts.This recruitment to the FuRMAS depends on previously successful cell adhesion. The establishment and analysis of modified D-Swip-1 in transgenic fly lines showed that both the EF-hand and the coiled-coil domains are required to recruit. We analysed many fusion-relevant mutants with respect to Swip-1 localization to place D-Swip into the cascade leading from cell adhesion to membrane merging. Our data to singles bar and blow mutants are of prime interest. Earlier EM studies hadshown that single bar mutants arrest myoblast fusion at the prefusion complex stage, which is characterized by accumulation of vesicles on both sides of the opposing membranes of FCs and FCMs, while blow mutants arrest thereafter, yet shortly before membrane fusion. DSwip-1 accumulates in single bar mutants at the site of fusion. Thus we propose that Drosophila Swip-1 might act among others during exocytosis of electron-dense vesicles. As a final step of fusion, the FCM needs to be incooperated into the growing myotube. Most of the musculature of Drosophila is newly formed during metamorphosis. The musculature of the testes is highly unusual as a multinucleated smooth musculature. We postulate that myoblast fusion in the embryo and during metamorphosis require common and different components for fusion. A protein interaction screen in yeast revealed a myosin heavy chain, Mhcl, being specifically expressed in FCs. Within the FuRMAS, Mhcl is connected to the cell adhesion protein Dumb founded by the adaptor protein Rolling pebbles. So we propose Mhcl interacts with the actin filaments to integrate FCMs into the growing myotube after the membranes merged.
Publications
- (2011). Drosophila Swiprosin-1/EFHD2 accumulates at the prefusion complex stage during Drosophila myoblast fusion. J Cell Sci 124, 3266-3278
Hornbruch-Freitag C, Griemert B, Buttgereit D and Renkawitz-Pohl R
- (2011). Role of the actin cytokeleton within FuRMAS during Drosophila myoblast fusion and first functionally conserved factors in vertebrates. In: Cell Fusions ed. L.-K.+L. Larsson, Springer pp 139–170
Önel SF, Dottermusch C, Sickmann A, Buttgereit D and Renkawitz-Pohl R
- (2012). Multinucleated smooth muscles and mononucleated as well as multinucleated striated muscles develop during the establishment of the male reproductive organs of Drosophila melanogaster. DevBiol 370, 86-97
Susic-Jung L, Hornbruch-Freitag C, Kuckwa J, Rexer KH, Lammel U and Renkawitz-Pohl R
(See online at https://doi.org/10.1016/j.ydbio.2012.07.022) - (2013). Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7. Exp Cell Res 319, 402-16
Bonn BR, Rudolf A, Hornbruch-Freitag C, Daum G, Kuckwa J, Kastl L, Buttgereit D, and Renkawitz-Pohl R
(See online at https://doi.org/10.1016/j.yexcr.2012.12.005) - (2014). Tethering membrane fusion: Common and different players in myoblast fusion and synaptic vesicle exocytosis. J Neurogenetics Jun 24:1-30
Önel S, Rust M, Jacob R and Renkawitz-Pohl R
(See online at https://doi.org/10.3109/01677063.2014.936014)
