Generation of a monoclonal antibody reactive to prefusion myocytes.

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion-incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell-cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.

Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells.

Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, beta-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

Meltrin beta/ADAM19 interacting with EphA4 in developing neural cells participates in formation of the neuromuscular junction.

BACKGROUND: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously. PRINCIPAL FINDINGS: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling. CONCLUSION: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Role of meltrin {alpha} (ADAM12) in obesity induced by high- fat diet.

Meltrin alpha is a member of the metalloprotease-disintegrin (ADAM) family. In this paper we demonstrate that meltrin alpha is involved in the development of white adipose tissue. Compared with wild-type mice, meltrin alpha(-/-) mice displayed moderate resistance to weight gain induced by a high-fat diet, mainly because of an impaired increase in the number of adipocytes. There was no obvious difference in adipocyte size between wild-type and meltrin alpha(-/-) mice, suggesting normal maturation of adipocytes of the latter under a high-fat diet. Embryonic fibroblasts and stromal-vascular cells lacking meltrin alpha exhibited impaired cell proliferation upon adipogenic stimulation, which was accompanied by moderate defects in adipose differentiation. Addition of culture medium conditioned with wild-type cells in an early phase of adipose differentiation did not restore the defects in the meltrin alpha(-/-) cells. These results uncover the involvement of meltrin alpha in the development of obesity and in adipogenic cell proliferation.

Lipid rafts identified as locations of ectodomain shedding mediated by Meltrin beta/ADAM19.

Meltrin beta (Mel beta, also called ADAM19) is a member of the ADAM (adisintegrin and metalloprotease) family, which are membrane-anchored glycoproteins that play crucial roles in various biological processes. Many intercellular signaling molecules are membrane-anchored proteins, which are proteolytically processed after becoming membrane-bound, to liberate their extracellular domains (ectodomain shedding). Genetic and biochemical studies have shown that some ADAMs participate in these events. We found previously that Mel beta can cleave the extracellular region of the membrane-anchored beta-exon-containing Neuregulin-1 (NRG beta1) protein, which is one of the main ligands for the neural ErbB receptor. Mel beta-deficient mice showed developmental defects in the nervous system. These observations raise the possibility that the NRG ectodomain shedding mediated by Mel beta is closely related to the neural development. Here we show that Mel beta-mediated ectodomain shedding of NRG beta1 takes place in the lipid rafts of neurons. The lipid rafts localization of Mel beta requires its membrane-anchoring region, and NRG beta1 ectodomain shedding is not enhanced if Mel beta cannot reach the lipid rafts. These results indicate that localization of Mel beta in lipid rafts is critical for its ectodomain shedding.

Essential roles of Meltrin beta (ADAM19) in heart development.

Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin beta/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin beta exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial-mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin beta is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin beta, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin beta-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin beta for proper heart development.

Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.

Meltrin beta mini, a new ADAM19 isoform lacking metalloprotease and disintegrin domains, induces morphological changes in neuronal cells.

Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system and other organs during embryogenesis. We report here an alternatively spliced isoform, meltrin beta mini, that lacks the prodomain, metalloprotease and disintegrin domains. A comparison of the cDNA and genomic sequences suggested the existence of a new exon. This isoform was detected in murine dorsal root ganglion and neuronal cell lines by RT-PCR. Overexpression of meltrin beta mini but not meltrin beta induced neurite outgrowth in neuronal cells. These studies suggest that the novel meltrin beta isoform has a distinct function related to neurogenesis.