CYTOPLASMIC REGULATION OF THE MOVEMENT OF E-CADHERIN ON THE FREE-CELLSURFACE AS STUDIED BY OPTICAL TWEEZERS AND SINGLE-PARTICLE TRACKING -CORRALLING AND TETHERING BY THE MEMBRANE SKELETON

The translational movement of E-cadherin, a calcium-dependent cell-cel l adhesion molecule in the plasma membrane in epithelial cells, and th e mechanism of its regulation were studied using single particle track ing (SPT) and optical tweezers (OT). The wild type (Wild) and three ty pes of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadheri ns that had deletions in the COOH terminus and lost the catenin-bindin g site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. Th e third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-ter minal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extrac ellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact r egion) was investigated. Catenin-minus and Short-tailed could be dragg ed an average of 1.1 and 1.8 mu m by OT (trapping force of 0.8 pN), an d exhibited average microscopic diffusion coefficients (D-micro) of 1. 2 x 10(-10) and 2.1 x 10(-10) cm(2)/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diff usion, The confinement area was 0.13 mu m(2) for Wild and Catenin-minu s, while that for Short-tailed was greater by a factor of four. In con trast, Fusion could be dragged an average of only 140 nm by OT. Averag e D-micro for Fusion measured by SPT was small (0.2 x 10(-10) cm(2)/s) . These results suggest that Fusion was bound to the cytoskeleton. Wil d consists of two populations; about half behaves like Catenin-minus, and the other half behaves like Fusion, It is concluded that the movem ents of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network o f the membrane skeleton (like Catenin-minus). The effective spring con stants of the membrane skeleton that contribute to the tethering and c orralling effects as measured by the dragging experiments were 30 and 5 pN/mu m, respectively, indicating a difference in the skeletal struc tures that produce these two effects.