Control of Myoblast Fusion by a Guanine Nucleotide Exchange Factor, Loner, and Its Effector ARF6

Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
Cell (Impact Factor: 32.24). 10/2003; 114(6):751-62. DOI: 10.1016/S0092-8674(03)00720-7
Source: PubMed


Myoblast fusion is essential for the formation and regeneration of skeletal muscle. In a genetic screen for regulators of muscle development in Drosophila, we discovered a gene encoding a guanine nucleotide exchange factor, called loner, which is required for myoblast fusion. Loner localizes to subcellular sites of fusion and acts downstream of cell surface fusion receptors by recruiting the small GTPase ARF6 and stimulating guanine nucleotide exchange. Accordingly, a dominant-negative ARF6 disrupts myoblast fusion in Drosophila embryos and in mammalian myoblasts in culture, mimicking the fusion defects caused by loss of Loner. Loner and ARF6, which also control the proper membrane localization of another small GTPase, Rac, are key components of a cellular apparatus required for myoblast fusion and muscle development. In muscle cells, this fusigenic mechanism is coupled to fusion receptors; in other fusion-competent cell types it may be triggered by different upstream signals.

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    • "The following mutant Drosophila melanogaster strains were used for mapping and characterization of the reported EMS alleles: Cg25C DTS-L3 and Cg25C b-9 ([30], gift from M. Mink, University of Szeged), LanA 9–32 ([98], gift from T. Volk, Weizmann Institute of Science), P{lacW}Cg25C k00405 , P{SUPor-P}LanB1 KG03456 , Mi{MIC}LanA MI02491 , Mi{ET1}LanA MB01129 , Mi{MIC}LanB2 MI03747 , Mi{ET1}prc MB03017 , P{PZ}vkg 01209 , P{PZ}wb 09437 , Df(2L)BSC110, Df(2L)BSC172, Df(2L)Exel7022, Df(2L)BSC233, Df(2L)ED12527 and about 180 additional deficiencies spanning chromosome 2 (all available from the Bloomington Stock Center at Indiana University, USA). For phenotypic analysis we used these additional reporter lines: Hand-GFP on chromosome 3 (HCH-GFP; [51]), Mhc-tau::GFP on chromosome X ([99]; obtained from F. Schnorrer, Max-Planck-Institute of Biochemistry) and the Flytrap GFP lines vkg G454 (vkg::GFP) and trol ZCL1973 (trol::GFP) ([58,59,100], obtained from L. Cooley, Yale University Medical School). For the analysis of pericardial cells in the absence of alary muscles, mutants of the desired allele were combined with the X-chromosomal mutation org-1 OJ487 [18] and crossed with males of the corresponding single mutant allele carrying Hand-GFP. "
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    ABSTRACT: Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.
    BMC Developmental Biology 06/2014; 14(1):26. DOI:10.1186/1471-213X-14-26 · 2.67 Impact Factor
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    • "We have gained insight into this process through a genetic screen designed to take advantage of the amenability of fly muscles to large-scale genetic analysis [42]. Previous large-scale screens, both in vivo and in primary cell culture, have established the fly as an excellent system in which to analyze muscle function [41] as well as specific aspects of muscle development such as myoblast fusion [55], myotube targeting [48], and muscle assembly [56]. Furthermore, these screens identified a large number of genes required for muscle function [41], [56]–[58]. "
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    ABSTRACT: Animal muscles must maintain their function while bearing substantial mechanical loads. How muscles withstand persistent mechanical strain is presently not well understood. The basic unit of muscle is the sarcomere, which is primarily composed of cytoskeletal proteins. We hypothesized that cytoskeletal protein turnover is required to maintain muscle function. Using the flight muscles of Drosophila melanogaster, we confirmed that the sarcomeric cytoskeleton undergoes turnover throughout adult life. To uncover which cytoskeletal components are required to maintain adult muscle function, we performed an RNAi-mediated knockdown screen targeting the entire fly cytoskeleton and associated proteins. Gene knockdown was restricted to adult flies and muscle function was analyzed with behavioural assays. Here we analyze the results of that screen and characterize the specific muscle maintenance role for several hits. The screen identified 46 genes required for muscle maintenance: 40 of which had no previously known role in this process. Bioinformatic analysis highlighted the structural sarcomeric proteins as a candidate group for further analysis. Detailed confocal and electron microscopic analysis showed that while muscle architecture was maintained after candidate gene knockdown, sarcomere length was disrupted. Specifically, we found that ongoing synthesis and turnover of the key sarcomere structural components Projectin, Myosin and Actin are required to maintain correct sarcomere length and thin filament length. Our results provide in vivo evidence of adult muscle protein turnover and uncover specific functional defects associated with reduced expression of a subset of cytoskeletal proteins in the adult animal.
    PLoS ONE 06/2014; 9(6):e99362. DOI:10.1371/journal.pone.0099362 · 3.23 Impact Factor
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    • "Second, Conu acts synergistically with the small GTPase Arf6 in causing overproliferation. Previous studies have shown that Arf6 promotes activation of Rac1 at the plasma membrane (Chen et al., 2003; D'Souza-Schorey and Chavrier, 2006; Koo et al., 2007; Bach et al., 2010). Consistent with the idea that Arf6 promotes Rac1 activity, coexpression of Arf6 strongly enhances the Rac1 eye phenotype. "
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    ABSTRACT: RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, the ERM protein Moesin, has the ability to bind to and organize cortical F-actin as well as the ability to regulate RhoA activity. ERM proteins have been shown previously to interact both with RhoGEFs and RhoGAPs, proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. We demonstrate that Moesin interacts with an unusual RhoGAP, Conundrum, and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, we show that cortically localized Conundrum can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conundrum's ability to promote growth also appears dependent on increased Rac activity. Our results reveal a molecular mechanism by which ERM proteins control RhoA activity, and suggest a novel linkage between the small GTPases RhoA and Rac in growth control.
    Molecular biology of the cell 03/2013; 24(9). DOI:10.1091/mbc.E12-11-0800 · 4.47 Impact Factor
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