Gene name - myoblast city
Cytological map position - 95B1--95C11
Function - docking protein
Symbol - mbc
Genetic map position -
Cellular location - cytoplasmic
|Recent literature||Biersmith, B., Wang, Z. H. and Geisbrecht, E. R. (2015). Fine-tuning of the actin cytoskeleton and cell adhesion during Drosophila development by guanine nucleotide exchange factors Myoblast city and Sponge. Genetics [Epub ahead of print]. PubMed ID: 25908317
The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to Engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes. Both in vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism to understand Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock-A) and Sponge (Spg; Dock-B). This study shows that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel (dv). Moreover, this study confirms the in vivo role of Mbc upstream of Rac and provides evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. These findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis.
|Duque, J. and Gorfinkiel, N. (2016). Integration of actomyosin contractility with cell-cell adhesion during dorsal closure. Development [Epub ahead of print]. PubMed ID: 27836966
This work combined genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure, during the morphogenesis of the amnioserosa, the main force-generating tissue during the process of Dorsal Closure in Drosophila. This work reveals that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. In contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, in amnioserosa cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through the control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.
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).
myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, it was established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or 'Docker'), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. The SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding (Balagopalan, 2006).
Dock180, the most extensively studied CDM family member, is a nonconventional GEF that facilitates GTP loading on Rac1. PXXP sites near its C terminus mediate interaction with the small SH2-SH3 adapter protein Crk, forming a complex that can increase GTP exchange on Rac1. These interactions form the basis for the Crk-Dock180-Rac1 signaling pathway. Dock180 has also been found in a trimolecular complex which, along with CrkII, contains the PH domain-containing protein Elmo. The primary role of Crk appears to be in recruiting these nonconventional GEFs to target sites at the cell membrane, thereby facilitating interaction with downstream effectors that regulate the cytoskeleton. Crk also appears to regulate the assembly and function of the Dock180/Elmo complex itself. Orthologs of these molecules have been identified in C. elegans, and genetic studies support a mechanism in which they function together in cell engulfment. In Drosophila, mutants with loss-of-function mutations in mbc, dominant negative and constitutively active forms of Rac1, and loss-of-function mutations in both rac1 and rac2 all exhibit defects in embryonic myoblast fusion. DCrk was isolated in a biochemical screen for molecules that interact strongly with the MBC C terminus, but efforts to determine its embryonic loss-of-function phenotype have been hindered by the presence of high levels of maternally provided transcript. Moreover, its location on the fourth chromosome effectively eliminates the possibility of generating germ line clones to uncover a role for DCrk during embryonic development. Notwithstanding these obstacles, or perhaps because of them, more recent studies have made use of RNA interference (RNAi) technology and insertional mutations to examine adult thorax closure, where maternally provided transcripts are not present to complicate the analysis. These studies have revealed modest genetic interactions between a DCrk insertional mutation, DCrk RNAi, D-Elmo RNAi, or mbc RNAi with loss-of-function alleles for Pvr, which encodes the Drosophila ortholog of the platelet-derived growth factor/vascular endothelial growth factor receptor. Genetic interactions between pathway members have also been observed in the Drosophila eye, where mutations in mbc suppress the effects of ectopic Rac1. Together, these observations support conservation of the Crk-CDM-Rac1 pathway in Drosophila (Balagopalan, 2006).
The studies presented herein were designed to examine MBC in the embryonic musculature. Along with the analysis of traditional EMS-induced point mutations in MBC, rescue of the loss-of-function embryonic muscle phenotype was examined. In this assay, a single copy of the mbc transgene is integrated into the genome, and expression is under the control of a single-copy muscle-specific promoter driving the yeast transcriptional activator GAL4. It was determined that the SH3 domain, the PtdIns(3,4,5)P3 binding DHR1 domain, and the Docker domain of MBC are all essential. The latter requirement is consistent with the rac1-associated myoblast fusion phenotype and supports the interpretation that, as in other systems, the Docker domain interacts with Rac1. Perhaps most interesting is the implication that phosphoinositol signaling is essential for myoblast fusion in a mechanism that appears to involve PtdIns(3,4,5)P3 binding to MBC (Balagopalan, 2006).
This study also addressed the in vivo relevance of the CDM-Crk biochemical association in Drosophila by examining the ability of mutant forms of MBC to rescue its loss-of-function muscle phenotype. Unexpectedly, in contrast to working models, the direct interaction of MBC with DCrk does not appear to be an essential part of the pathway through which MBC regulates myoblast fusion. Biochemical and yeast two-hybrid interactions confirm that the Crk-binding sites in MBC are active and are the only regions of MBC to directly interact with Crk. Nevertheless, MBC appears to be fully functional and present at the membrane despite the absence of these sites. This finding is reminiscent of results in cultured cells transiently transfected with Dock180, in which it cooperates with Elmo to stimulate migration in a transwell assay in the absence of the Crk-binding sites. The results extend this analysis to MBC in the musculature of the intact embryo and establish that no other regions of MBC directly interact with DCrk in the absence of the C terminus (Balagopalan, 2006).
It remains to be determined how MBC is localized to the membrane, assuming that this is an important step in the pathway. Notably, simple membrane targeting of full-length MBC via a myristoylation signal does not rescue myoblast fusion. It is a formal possibility that DCrk directs membrane association of MBC through a novel mechanism that does not involve its direct binding to MBC or that different domains of MBC function redundantly with DCrk in this capacity. In mammalian cells, the SH3 domain of Dock180 can target it to the membrane through its interaction with Elmo and, in turn, Elmo's interaction with RhoG. In addition, interaction of the highly conserved DHR1 domain with PtdIns(3,4,5)P3 directs Crk-independent membrane localization of Dock180. By comparison, MBC is present at the membrane when the PtdIns(3,4,5)P3 binding domain is deleted and when mutations rendering the SH3 domain nonfunctional are present. Thus, none of these domains are exclusively essential for membrane targeting of MBC in the somatic mesoderm. Despite the possibility that they act redundantly with DCrk in directing membrane localization of MBC, however, the SH3, DHR1, and Docker domains clearly play essential roles independent of this process in the ability of MBC to direct myoblast fusion (Balagopalan, 2006).
Interestingly, a role in recruitment of MBC to target sites at the membrane in the embryonic musculature has been suggested for the founder myoblast-specific Ants/Rols protein. This protein links an N terminus-containing fragment of MBC to the cytoplasmic domain of the founder-specific cell adhesion molecule Duf/Kirre. Studies have not yet addressed whether the domains that direct these interactions are essential or whether similar interactions occur between Ants/Rols and IrreC/rst, which functions redundantly with Duf/Kirre in the musculature. Moreover, the data suggest that MBC expression is also required in the fusion-competent cells, which lack Ants/Rols. One might anticipate the presence of a molecule similar to Ants/Rols that recruits MBC to the cytoplasmic domain of the SNS cell adhesion molecule in these myoblasts. In a variation of this model, PXXP sites in the SNS cytoplasmic domain may interact directly with the SH3 domain of MBC, thereby recruiting it to the membrane of the fusion-competent cells. In summary, it is becoming increasingly apparent that the well-characterized interactions of CDM proteins with Crk, Elmo, and Rac1 represent only a subset of their potential partners. Future studies will likely reveal a larger spectrum of proteins through which they act (Balagopalan, 2006).
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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