Members of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors function in diverse processes that include cell migration and myoblast fusion. Previous studies have shown that the SH3, DHR1 and DHR2 domains of Myoblast city (MBC) are essential for it to direct myoblast fusion in the Drosophila embryo, while the conserved DCrk-binding proline rich region is expendable. Herein, we describe the isolation of Drosophila ELMO/CED-12, an approximately 82 kDa protein with a pleckstrin homology (PH) and proline-rich domain, by interaction with the MBC SH3 domain. Mass spectrometry confirms the presence of an MBC/ELMO complex within the embryonic musculature at the time of myoblast fusion and embryos maternally and/or zygotically mutant for elmo exhibit defects in myoblast fusion. Overexpression of MBC and ELMO in the embryonic mesoderm causes defects in myoblast fusion reminiscent of those seen with constitutively-activated Rac1, supporting the previous finding that both the absence of and an excess of Rac activity are deleterious to myoblast fusion. Overexpression of MBC and ELMO/CED-12 in the eye causes perturbations in ommatidial organization that are suppressed by mutations in Rac1 and Rac2, demonstrating genetically that MBC and ELMO/CED-12 cooperate to activate these small GTPases in Drosophila.

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH ) was cloned and sequenced. The relA VCH gene encodes a 738-aminoacid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ⌬relA::kan allele was generated by replacing ϳ31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.Vibrio cholerae is a facultative anaerobic gram-negative bacterium and the causative agent of the severe diarrheal disease cholera. In addition to residing temporarily in the intestinal lumen of humans during the diseased state, V. cholerae has its natural niche in the aquatic environment, residing in the freeliving aquatic flora found in estuarine areas and in association with crustaceans and mollusks (25). The strains of V. cholerae that cause epidemic cholera belong to serogroups O1 and O139 (3, 4, 28, 41, 50). The O1 serogroup is again divided into two biotypes, classical and El Tor (28). Strains other than O1 and O139 are known as non-O1/non-O139 vibrios.A pathogen in its natural environment and host-associated state is subjected to a plethora of stresses, such as fluctuations in pH, salinity, osmolarity, oxygen tension, temperature, and nutritional availability. These offer selective pressure to a bacterium, eliciting various adaptive responses for its survival. The adaptive response to nutritional stre...

Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion.

We have been unable to reproduce the data in Figure 1H, reporting that the myoblast fusion defect in mbc mutant embryos is rescued by expression of constitutively activated Rac1. Although we are unable to confirm the source of the error, all stocks have been reconfirmed and all other results in the original publication independently confirmed by two of the authors.

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process [1][2][3][4]. With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references. Founder Cells and FCMs: Precursors of the Somatic MusculatureBoth founder cells and FCMs arise from small clusters of cells within the mesoderm. Through Notch-mediated lateral inhibition, a single cell within each cluster is selected. In at least some cases, this cell undergoes an additional cell division to give rise to two founder cells or a founder cell and another cell type [5]. The FCMs arise from the same clusters of mesodermal cells, deriving from those that remain after Notch-mediated founder cell selection. As in segregation of neuroblasts and epidermal cells in the ectoderm, all cells in a cluster become founder myoblasts in the absence of Notch [6]. The specific identity of each founder cell is then established by expression of a specific combination of "muscle identity genes", and the absence of a particular muscle identity gene results in the loss of specific muscle fibers [5]. The combination of muscle identity genes also determines the ultimate morphology and size of the final muscle fiber, as evidenced in recent studies by the demonstration that misexpression of identity genes alters the extent of fusion and subsequent behavior of the resulting muscle fiber [7]. It therefore seems apparent that founder cells