BIOLOGICAL OVERVIEW

Alleles of myoblast city were recovered in a screen designed to make mutations in the 95 region of the third chromosome. All known mbc alleles are recessive and cause fatal embryonic defects. Mutant embryos lie motionless in the vitelline membrane and fail to hatch. Examination of the mbc mutant embryos with a polarized light microscope shows a striking lack of differentiated muscle (Rushton, 1995). Mbc is one of the first proteins in Drosophila to be identified as being essential for myoblast fusion. It is expressed in a broad range of tissues throughout embryonic development, including the presumptive musculature and epidermal cells involved in the process of dorsal closure. Consistent with its expression pattern, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization as well as myoblast fusion. These abnormalities are similar to those for the small GTPase Drac1, and suggest that mbc functions in the epidermis in the same pathway as Rac1, and that this pathway is used in the mesoderm for events leading to myoblast fusion. Mbc has striking homology to DOCK180, a human gene that was identified on the basis of its interaction with the small adapter protein Crk. Genes identified in several genome projects suggest that DOCK180 and Mbc define a new gene family (Erickson, 1997).

Because mbc is expressed early in the ectoderm and persists in the epidermis, mbc mutant embryos were examined for epidermal defects. Using Fasciclin III as a marker, mbc mutant embryos were unable to complete the process of dorsal closure. Contractile filaments formed from actin and myosin are thought to provide the driving force for dorsal closure. Consistent with this suggestion, the absence of nonmuscle myosin in zipper mutant embryos is likely to be responsible for their failure to complete this process. Similarly, overexpression of a form of Drosophila Rac1 that disrupts both actin and nonmuscle myosin accumulation at the leading edge of the migrating epidermis also inhibits dorsal closure. Finally, the dorsal closure defects observed in mbc mutants are accompanied by reduced detection of filamentous actin. These results implicate mbc in cytoskeletal organization and dorsal closure and suggest that it may function in the same pathway as Rac1 (Erickson, 1997 and references).

The most apparent mesodermal defect in embryos mutant for the mbc gene is the inability of myoblasts to fuse into muscle fibers, suggesting a role for mbc in the progression of cells from myoblasts to myotubes. This multistep process has been divided into several stages and includes the acquisition of fusion competence, a time-dependent behavior that may be related to withdrawal from the cell cycle, myoblast adhesion, and plasma membrane union (Erickson, 1997 and references).

At least two features of the mbc-encoded protein seem somewhat inconsistent with a role in either cell adhesion or membrane fusion itself. (1) MBC does not have sequence features reminiscent of cell adhesion molecules and appears to be present throughout the cytoplasm rather than membrane bound. (2) Both Mbc and its structural homolog, DOCK180, are expressed in a wide range of tissues that do not fuse. It is thought that the function of Mbc in the mesoderm is analogous to its role in the epidermis and that it functions as an essential intermediate in a signal transduction cascade that also includes the small GTPase Rac1. This pathway could involve tyrosine phosphorylation of complexes that directly modulate events in the cytoskeleton requiring proteins that include Mbc. Alternatively, Mbc may function in signal transduction to the nucleus via the Ras and MAP kinase pathway and may affect the cytoskeleton only indirectly. Interestingly, while vertebrate studies have not revealed a specific requirement for focal adhesions in myogenesis, they have implicated extracellular matrix components that stimulate focal adhesions, such as fibronectin, in myogenic differentiation. Additional studies in vertebrates support a role for the cytoskeleton in myoblast fusion. Myoblast fusion is severely limited in the presence of cytochalasin B, an alkaloid that interferes with the assembly of actin filaments. While the role of the cytoskeleton in myoblast fusion remains unclear, it may be involved in the formation of lipid-rich domains within the cell membrane that create sites for membrane-membrane fusion (Erickson, 1997 and references). Alternatively, actin filaments may be required for the formation or organization of vesicles (prefusion complexes) that have been observed under the plasma membrane just before fusion of both vertebrate and Drosophila myoblasts (Doberstein, 1997).

The behavior of these vesicular complexes is unprecedented, with multiple pairs of vesicles from different cells aligning with each other across a pair of plasma membranes. It is believed that the paired vesicles are of prime importance to later steps in the myoblast fusion process since mbc myoblasts (which have no prefusion complexes) also lack electron-dense plaques (normally seen at the site of fusion), and do not align or fuse. Vesicles with electron dense material along their cytoplasmic surfaces have been reported in primary cultures of quail myoblasts and in a muscle cell line. The pairing behavior and the electron dense material between cells were not described in either case. The quail vesicles were shown to fuse with the plasma membrane, and in at least one case, a pair of those vesicles in apposed cells were shown in the act of fusing simultaneously with their respective plasma membranes. It is unclear whether the vesicles described in these previous studies are analogs of the paired vesicles see in Drosophila. Prefusion complexes are present in blown fuse embryos (blown fuse mutants are also defective in myoblast fusion, see below), and absent in mbc embryos (which are defective in recognition and/or adhesion). It therefore seems clear that the prefusion complex forms only after the recognition of (and perhaps adhesion to) an appropriate fusion target cell (Doberstein, 1997).

What is the function of the paired vesicles? The paired vesicles may contain the essential components of the fusion apparatus destined for the plasma membrane, particularly the electron-dense material making up the plaques that sometimes appear in later steps of the fusion process. Alternatively, the paired vesicles might have a specific mechanistic role in the fusion process beyond simple delivery of components to the apposed plasma membranes. A third possibility is that the vesicles might have a role in the recognition and/or attachment phase of the process. If the recognition phase were aborted by lack of vesicles, no further progression to the attachment phase would be expected. The 1:1 pairing of vesicles in different cells across their apposed plasma membranes suggests some hypotheses for the function of these organelles. If the vesicles have a mechanistic role in later fusion events, the exact geometry of paired vesicles in the prefusion complex relative to the plasma membranes and each other might be of prime importance. If the paired vesicles have a simple role of delivering fusion components to the plasma membranes, the pairing might serve two functions: (1) docking the vesicles to a prefusion complex would serve to restrict the plasma membrane distribution of potentially fusogenic macromolecules to the small area where fusion is necessary and not to regions where fusion would be inappropriate, and (2) pairing of vesicles might enable a strict 1:1 ratio of molecules essential for fusion in the fusing region of each cell (Doberstein, 1997).

In either case, the presence of paired vesicles and the apparent symmetry of the prefusion complex strongly argues for a bidirectional function of the fusion event, that is, that there is not a "donor/receiver" relationship between the fusing cells once the prefusion complex is formed. It is therefore hypothesized that the protein and lipid composition of the two plasma membranes in the fusing areas are nearly identical, and that the mechanics of the fusion process take place in a symmetrical fashion. This theoretical homotypic fusion is quite different from heterotypic fusion, for example, infection of cells by enveloped viruses, in which the viral membrane contains different components of the fusion process, that is to say, different from those in the membrane of the target cell. The apparent bidirectional nature of the fusion process also implies that the fusing myoblasts are able to identify appropriate targets for fusion (i.e., myotubes or muscle pioneer cells) before the formation of the prefusion complex. This concept is supported by the absence of prefusion complexes in mbc mutants, which appear to be defective in recognition and/or adhesion to fusion targets (Doberstein, 1997).

The absence of paired vesicles in mbc mutant embryos may be a consequence of defects in the actin cytoskeleton (Doberstein, 1997). Doberstein places mbc upstream of a constitutively active form of Drac1. As discussed by these authors, however, the analysis of Drac1 is presently limited to targeted expression of altered forms of the protein and is problematic in the absence of a loss-of-function mutation. It may also reflect a second role for Rac1 in myoblast fusion, not inconsistent with the suggestion that GTPases may act downstream of focal adhesions. One intriguing possibility consistent with the data of both Erickson and Doberstein is an early requirement for activated Rac1, perhaps to facilitate recruitment of paired vesicles to the membrane via the cytoskeleton, followed by an equally important requirement for Rac1 inactivation later, before fusion. One final issue is that genetic studies have not yet revealed a role for integrin subunits, one of the major components of vertebrate focal adhesions, in myoblast fusion. The larval body wall muscles in embryos mutant for the major integrin subunits, betaPS, alphaPS1, and alphaPS2, do not appear to exhibit defects in fusion. However, the number and alternatively spliced forms of integrins identified in Drosophila have continued to increase, and family members that play other roles in myogenesis may yet be isolated. Thus, greater knowledge of GTPases and integrins and the identification of Drosophila homologs with components of vertebrate focal adhesions are likely to refine ideas of Mbc's role in myoblast fusion (Erickson, 1997).

Doberstein (1997) implicates a second protein, Blown Fuse, in the process of myoblast fusion, and proposes an elaborate model for fusion. Initially, myoblasts identify and adhere to fusion targets, either muscle pioneer cells or existing myotubes. This step may very well involve multiple separate stages, including chemoattraction of myoblasts to fusion targets, cell-cell communication for identification of target cells, and cell adhesion. The phenotype of mbc is consistent with a block somewhere in the process before cell adhesion. Pairs of cells that have correctly identified appropriate fusion targets then set up prefusion complexes at contact points where fusion will eventually begin. These complexes include paired vesicles and their associated electron dense material. The myoblasts become elongated, and align themselves along their long axes. Defects in the blown fuse gene stop the process before alignment takes place. What might the function of the Blown Fuse protein be in normal myoblasts? It is hypothesized that Blown Fuse is required for the normal function of the prefusion complex, even though it is not an integral component of that complex. Blown Fuse might have an enzymatic activity necessary for prefusion complex function. The structure of the prefusion complex taken along with the relative scarcity of plaques suggests that paired vesicles and other complex components are accumulated at contact sites and remain quiescent for a relatively long period of time before dispersing by forming a plaque. Perhaps a signal transduction cascade must be activated before the complex can complete its normal function, with Blown Fuse being an essential part of that cascade. A third possibility is that the Blown Fuse protein is part of a checkpoint system that allows progress through the fusion process only after proper function of the prefusion complex, and that later steps are inhibited due to improper functioning of the checkpoint system. After an unknown signal, the prefusion complex resolves into a short-lived electron-dense plaque. It is not clear from this work whether alignment must take place before the plaque stage or whether the two events happen independent of one another. The rolling stone mutation, also exhibiting a fusion phenotype, causes aberrant accumulation of plaques in stage 13 embryos, although the plasma membranes are able to become closely apposed as seen when the accumulated plaques disperse by stage 14. Next, fusion pores form, making the cytoplasm of the fusing cells continuous. Dominantly active gain-of-function Rac1 blocks the formation of the pores. The pores expand and the plasma membrane breaks down into smooth sacs of membrane. With time, these sacs become rounder in profile and eventually are accumulated in groups of clear, irregularly shaped vesicles before recycling or disposal. It is concluded that myoblast fusion is a complex process involving a novel vesicular complex and a number of dedicated gene products (Doberstein, 1997).

PROTEIN STRUCTURE

Amino Acids - 1970

Structural Domains

Data-base homology comparisons using BLAST aligns the Mbc protein with DOCK180, a human protein of 1,866 amino acids. DOCK180 was isolated on the basis of an interaction with Crk, a small adapter protein consisting mainly of SH2 and SH3 domains. Mbc and DOCK180 have significant homology throughout their entire length. In particular, DOCK180 contains a putative SH3 domain that proceeds from amino acids 11-71 and includes the three essential SH3 consensus residues. These three residues, along with several others within this domain, are identical in Mbc. DOCK180 contains two copies of the Crk-binding consensus site PPxLPxK, while Mbc has one exact and one slightly divergent copy of this consensus site. By contrast, the putative ATP-binding site is not conserved. Several additional blocks of homology are present, notably a region in which 24 of 27 amino acids are identical (residues 1566-1592 of Mbc). Subsequent BLAST searches also reveal two ORFs with extensive homology to Mbc and DOCK180. The first ORF is from a human myeloid cell line, and the second is from the Caenorhabditis elegans genome project. The predicted myeloblast protein is highly homologous to both Mbc and DOCK180, while the predicted C. elegans protein is more divergent. Partial sequence from a mouse gene suggests the existence of a murine homologue as well (Erickson, 1997).

date revised: 16 November 98

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