A role for the Ca 2+-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

Abstract

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca 2+-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca 2+-independent (CI) 2 2 Abbreviations used: CI, Ca 2+-independent; CD, Ca 2+-dependent; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; EE, embryo extract; EGTA, ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; HHBSS, 10 m M Hepes-buffered Hanks' balanced salt solution; NP-40, Nonidet P-40; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; TE cells, cells exposed to 0.01% trypsin/ 2.5 m M EDTA; TC cells, cells exposed to 0.01% trypsin/2.5 m M Ca 2+; LTE cells, cells exposed to 0.0005% trypsin/2.5 m M EDTA. and Ca 2+ dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca 2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.