ABSTRACT
Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. In this study we found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. We demonstrate that the schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, we show that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, we employed epistasis experiments in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. We propose that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.
ABSTRACT
Fibromyalgia is characterized by widespread musculoskeletal pain, fatigue, and depression. The aim was to analyze potential mitochondrial dysfunction or autophagy in mice after exposure to intermittent cold stress (ICS). Muscle and liver specimens were obtained from 36 mice. Lactate dehydrogenase (LDH) activity was measured. Microtubule-associated protein light chain 3 (MAP1LC3B) and glycogen content were determined histologically; muscle ultrastructure by electron microscopy. Mitochondrial- and autophagy-related markers were analyzed by RT-qPCR and Western blotting. ATP level, cytotoxicity, and caspase 3 activity were measured in murine C2C12 myoblasts after ICS exposure. Coenzyme Q10B (COQ10B) transcript was up-regulated in limb muscle of ICS mice, whereas its protein content was stable. Cytochrome C oxidase 4 (COX4I1) and LDH activity increased in limb muscle of male ICS mice. Glycogen content was lower in muscle and liver tissue of male ICS mice. Electron micrographs of ICS mice specimens showed mitochondrial damage and autophagic vesicles. A significant up-regulation of autophagic transcripts of MAP1LC3B and BECLIN 1 (BECN1) was observed. Map1lc3b protein showed an aggregated distribution in ICS mice and SqSTM1/p62 (p62) protein level was stable. Furthermore, ATP level and caspase activity, detected as apoptotic marker, were significantly lowered after ICS exposure in differentiated C2C12 myoblasts. The present study shows that ICS mice are characterized by mitochondrial dysfunction, autophagic processes, and metabolic alterations. Further investigations could dissect autophagy process in the proposed model and link these mechanisms to potential therapeutic options for fibromyalgia.
ABSTRACT
BACKGROUND: The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. RESULTS: Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. CONCLUSIONS: Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.
