Role of NCAM, cadherins, and microfilaments in cell-cell contact formation in TM4 immature mouse sertoli cells

To determine events that lead to the formation of intercellular contacts, we examined the spatial and temporal distribution of NCAM, cadherins, and F-actin in TM4 cells by immunofluorescence and laser scanning confocal microscopy. TM4 cells exhibited epithelioid characteristics and formed large overlapping lamella-like cell-cell contacts that contained a high concentration of NCAM. NCAM-rich lamellae formed from smaller NCAM patches at the ends of filopodia-like contacts between adjacent cells. Cadherins, as visualized by a pan-cadherin antibody, were present in a pattern distinctly different from that of NCAM. Although in filopodia-like contacts, both cadherins and NCAM were often concentrated at filopodial tips, in the larger lamella-like contacts that developed later, cadherins were located in an irregular punctate pattern only at the distal and more apical margins of the slanted NCAM-rich contact regions. Patterns of NCAM and microfilament (MF) bundle distribution were distinctly different, suggesting that the ends of these MF bundles were not physically linked to NCAM. By contrast, cadherins were concentrated at the ends of MF bundles at all stages of contact formation examined. Interestingly, this association of cadherins with MF bundles was mostly seen at the edge of the overlapping processes. In the lower cell process, MF bundles at the contact site were often arranged in random fashion, indicating an asymmetric distribution of MF in the junctional region. However, N-cadherin was enriched only at sites where MF bundles from both the upper and lower cell processes were aligned and terminated at the junctional membrane. Thus the organization of the actin cytoskeleton at cell-cell contact sites is influenced by the differential localization of different cadherins. These data also suggest that different mechanisms are involved in the accumulation of NCAM and cadherins in cell-cell contact regions.