N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin–dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin–mediated signals involved in myogenesis, we investigated whether N-cadherin–dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin–dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin–mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for β-catenin accumulation at cell–cell contact sites. We propose that cell–cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin-dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin-dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.

Cadherins are transmembrane glycoproteins that mediate Ca2؉ -dependent homophilic cell-cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin-mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin-dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin-dependent contact formation, and myoblast fusion. We conclude that M-cadherin-dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion. INTRODUCTIONDuring skeletal muscle development mesodermal precursor cells give rise to committed myoblasts that, after proliferation and migration to the appropriate sites in the embryo, exit the cell cycle, express muscle-specific genes, and fuse into multinucleated myofibers that mature to form multinucleated muscle fibers (Taylor, 2002). Although myoblast fusion is important both during embryonic development and in the maintenance and repair of adult muscles, the mechanisms regulating this process are largely unknown. Myoblast fusion is a multistep process that entails initial recognition and adhesion between myoblasts, their alignment, and finally membrane breakdown and fusion (Doberstein et al., 1997). This process is regulated, at different levels, by a variety of proteins, such as transcription factors or extracellular signaling molecules, including diffusible factors, components of the extracellular matrix, and proteins involved in cell-cell contact (Krauss et al., 2005).In this later group, M-cadherin plays a prominent role. M-cadherin belongs to the cadherin family of Ca 2ϩ -dependent adhesion molecules. Its N-terminal extracellular domain mediates homophilic binding, while the cytoplasmic tail interacts with catenins and is linked to the actin cytoskeleton, thus, coupling the ectodomain interactions to the dynamic intracellular tensile forces (Wheelock and Johnson, 2003b). M-cadherin is found predominantly in developing skeletal muscles and is highly expressed during secondary myogenesis. In mature skeletal muscle, M-cadherin is detectable in satellite cells and on the sarcolemma of myofibers underlying satellite cells (Moore and Walsh, 1993;Rose et al., 1994;Cifuentes-Diaz et al., 1995). M-cadherin is also found at neuromuscular junctions, intramuscular nerves, and in two regions of the CNS, namely the spinal cord and the cerebellum (Cifuentes-Diaz et al., 1996;Bahjaoui-Bouhaddi et al., 1997). M-cadherin-deficient mice do not show defects in skeletal muscle development, probably because of compensation by other cadh...

The Rho family of GTP-binding proteins plays critical roles during myogenesis induction. To elucidate their role later during myogenesis, we have analyzed RhoA function during myoblast fusion into myotubes. We find that RhoA activity is rapidly and transiently increased when cells are shifted into differentiation medium and then is decreased until myoblast fusion. RhoA activity must be down-regulated to allow fusion, because expression of a constitutively active form of RhoA (RhoAV14) inhibits this process. RhoAV14 perturbs the expression and localization of M-cadherin, a member of the Ca 2؉ -dependent cell-cell adhesion molecule family that has an essential role in skeletal muscle cell differentiation. This mutant does not affect N-cadherin and other proteins involved in myoblast fusion, ␤1-integrin and ADAM12. Active RhoA induces the entry of M-cadherin into a degradative pathway and thus decreases its stability in correlation with the monoubiquitination of M-cadherin. Moreover, p120 catenin association with M-cadherin is decreased in RhoAV14-expressing cells, which is partially reverted by the inhibition of the RhoA effector Rho-associated kinase ROCK. ROCK inhibition also restores M-cadherin accumulation at the cell-cell contact sites. We propose that the sustained activation of the RhoA pathway inhibits myoblast fusion through the regulation of p120 activity, which controls cadherin internalization and degradation.