Genetic Analysis of Myoblast Fusion: blown fuse Is Required for Progression Beyond the Prefusion Complex

Abstract

The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of “paired vesicles.” These pairs of vesicles from different cells align with each other across apposed plasma membranes. This prefusion complex resolves into dense membrane plaques between apposed cells; these cells then establish cytoplasmic continuity by fusion of small areas of plasma membrane followed by vesiculation of apposed membranes. Different steps in this process are specifically blocked by mutations in four genes required for myoblast fusion. One of these genes, blown fuse, encodes a novel cytoplasmic protein expressed in unfused myoblasts that is essential for progression beyond the prefusion complex stage.

FuRMAS: triggering myoblast fusion in Drosophila

“…For example, Beckett and Baylies (2007) carefully counted the number of nuclei found in distinct muscle precursors in many known fusion mutants. Among these, only in myoblast city (mbc) mutants (Rushton et al, 1995;Erickson et al, 1997;Galletta et al, 1999) no fusion occurs at all as has been reported previously (Doberstein et al, 1997;Schröter et al, 2004Schröter et al, , 2006Menon et al, 2005). Interestingly, in agreement with previously published data, Beckett and Baylies (2007) found different numbers of nuclei in blow, kette, schizo/loner, mbc, and rols mutants as well as states of fusion between the segments of the individual mutants and concluded that there are no genetic differences between the two-phases of myoblast fusion, but rather only a delay in the fusion process itself.…”

“…A systematic analysis of these vesicles in wild-type embryos suggests that this complex consists of an average of 1.4 vesicles per complex in an individual section (Estrada et al, 2007). Kim et al (2007) also described variable numbers of vesicles, which were also not all paired, a finding that is supported by Doberstein et al (1997). These observations raise two key questions: (1) Why are the vesicle numbers so different?…”

“…A further question concerns the number and spatial distribution of paired vesicles at the opposed membranes. Here, the serial sections of Doberstein et al (1997) are highly informative since a total of 45 pairs of vesicles were found in six serial sections (each 100 nm), and these vesicles are distributed among four cells, one probably being a fc/growing myotube, and the three others probably being fcms (Fig. 3A).…”

“…2A). Genetic (Rau et al, 2001;Massarwa et al, 2007;Berger et al, 2008) as well as ultrastructural (Doberstein et al, 1997;Schröter et al, 2006) analyses led to the proposal that several distinct genetic players act during the two temporal fusion phases. This led to a two-phase model of myoblast fusion having common and different fusion-relevant proteins and signalling factors that are essential during the two-phase model.…”

“…2C). Doberstein et al (1997) suggested that these vesicles form a prefusion complex that can consist of up to 15 vesicles (40 nm in diameter) per contact site. From the Golgi to the membrane, they might be transported by F-actin, which disintegrates after prefusion complex formation (Kim et al, 2007).…”