Rac1 and RhoA GTPases have antagonistic functions during N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts

Abstract

Background information. N-cadherin, a member of the Ca 2+ -dependent cell-cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N-cadherin-dependent cell-cell contacts in myoblasts remain unexplored.Results. In the present study, we show that N-cadherin-dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N-cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N-cadherin-dependent cell contacts, characterized by the immobilization of a pool of N-cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F-actin filaments. We also demonstrated that the formation of N-cadherin-dependent cell-cell contacts requires a co-ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N-cadherin-dependent cell-cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N-cadherin-dependent cell contact formation, since, via ROCK (Rho-associated kinase) signalling and myosin 2 activation, it allows the stabilization of N-cadherin at the cell-cell contact sites. Conclusions.We have shown that Rac1 and RhoA have opposite effects on N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.

Cadherin Point Mutations Alter Cell Sorting and Modulate GTPase Signaling

“…Interestingly, the S78A mutant activated similar RhoA-GTP levels to N-CHO, but with slower kinetics. This inverse correlation between Rac1 and RhoA agrees with reports suggesting that RhoA and Rac1 have antagonistic effects (Comunale et al, 2007;Wildenberg et al, 2006).…”

“…Because we compared proteins with the same overall backbone and cytoplasmic domain, this correlation might not apply for general comparisons across cadherin subtypes due to possible differences in their interactions with GTPases (Anastasiadis et al, 2000;Boulter et al, 2010). Ccadherin and E-cadherin activate Rac1 (Noren et al, 2003;Yap and Kovacs, 2003), but N-cadherin ligation triggers RhoA activation (Charrasse et al, 2002;Comunale et al, 2007;Marrs et al, 2009;Taulet et al, 2009). Whether the latter is due to low N-cadherin affinity and Rac1/RhoA antagonism (Boulter et al, 2010;Burridge and Doughman, 2006;Comunale et al, 2007;Wildenberg et al, 2006), or to differences in GTPase interactions with N-cadherin complexes (Anastasiadis et al, 2000) remains to be determined.…”

EMT, the cytoskeleton, and cancer cell invasion

“…Fer then phosphorylates and activates cortactin, thereby inducing actin remodeling, and increasing the mobility of N-cadherin molecules to extend the adhesion zone and, finally, to promote the formation of stable cell-cell adhesion. In line with these observations, N-cadherin is found to be localized in the lamellipodia of adjacent, contacting myoblasts [84]. Fer also phosphorylates the phosphatase protein tyrosine phosphatase 1B (PTP1B) and promotes its binding to Ncadherin.…”

“…Lamellipodia interact and attach to their environment via different adhesion molecules, including integrins and cadherins [84,178]. Also, CD44, the hyaluronan receptor was identified to be in a complex with the protease MT1-MMP in lamellipodial protrusions [179].…”

Flotillin micro-domains stabilize Cadherins at cell-cell junctions

“…In various cell types, newly formed cell-cell contacts can be differentiated from established mature ones (Comunale et al, 2007;Affentranger et al, 2011;Taguchi et al, 2011). In Caco-2 cells, in which these two stages are easily distinguishable, flotillins were barely detectable at new CCJs, whereas they accumulated at mature CCJs (supplementary material Fig.…”