Akira Nagafuchi

Expressed recombinant cadherins mediate cell sorting in model systems

Cadherins are cell-surface glycoproteins responsible for Ca2+-dependent cell-to-cell adhesion. E- or P-cadherin was transfected into L cells, which normally have little cadherin activity, and cellular aggregation of the resulting transfectants was observed to be a function of the cadherin molecule expressed. Transfected cells preferentially adhered to cells expressing the same cadherin subclass. Furthermore, in reconstituted embryonic lung tissue, E-cadherin-expressing L cells were associated with epithelial tubules expressing E-cadherin, while untransfected L cells associated with mesenchymal cells. These results provide the first direct evidence that the differential expression of cadherins can play a role in cell sorting in heterogeneous cell populations.

The 102 kd cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression

The E-cadherin cell adhesion molecule is associated with cytoplasmic polypeptides, and this association is essential for its cell-binding function. Using isolated adherens junctions of the liver, we purified a 102 kd protein that can associate with E-cadherin (CAP102) and isolated cDNAs encoding this protein. Sequence analysis of the cDNAs revealed that this protein has a similarity to vinculin. L cells not expressing endogenous cadherin express the mRNA for CAP102 but have only a trace amount of CAP102 protein. Introducing exogenous E-cadherin into these cells, however, induced a high expression of CAP102 protein without affecting the amount of its mRNA, suggesting that there is a posttranscriptional regulatory mechanism for this molecule. The same effect was observed by introducing N- or P-cadherin into L cells.

Molecular linkage between cadherins and actin filaments in cell—cell adherens junctions

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.

Ca2+-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions

In multicellular organisms, various compositionally distinct fluid compartments are established by epithelial and endothelial cellular sheets. For these cells to function as barriers, tight junctions (TJs) are considered to create a primary barrier for the diffusion of solutes through the paracellular pathway [1] [2] [3]. In ultrathin sections viewed under electron microscopy, TJs appear as a series of apparent fusions, involving the outer leaflets of plasma membranes of adjacent cells, to form the so-called kissing points of TJs, where the intercellular space is completely obliterated [4]. Claudins are a family of 16 proteins whose members have been identified as major integral membrane proteins localized exclusively at TJs [5] [6] [7] [8]. It remains unclear, however, whether claudins have the cell-adhesion activity that would explain the unusual intercellular adhesion at TJs. Using mouse L-fibroblast transfectants expressing various amounts of claudin-1, -2 or -3, we found that these claudins possess Ca(2+)-independent cell-adhesion activity. Using ultrathin-section electron microscopy, we observed many kissing points of TJs between adjacent transfectants. Furthermore, the cell-adhesion activity of occludin, another integral membrane protein localized at TJs [9] [10] [11], was negligible when compared with that of claudins. Thus, claudins are responsible for TJ-specific obliteration of the intercellular space.

E-cadherin functions as a cis-dimer at the cell-cell adhesive interface in vivo.

310 nature structural biology • volume 6 number 4 • april1999 Cadherins are homophilic calciumdependent cell–cell adhesion molecules involved in specific cell recognition1,2. Recent studies of the three-dimensional structure of cadherin3,4 suggest that the cadherin extracellular domain forms a parallel dimer. However, to date no evidence has been reported for the existence of this parallel dimer in vivo and details of how this dimer formation is regulated are still fragmentary5,6. Here, using chemical crosslinking, we confirmed that E-cadherin forms a parallel (or ‘cis’) dimer (consisting of two molecules on the same cell surface) in the presence of calcium in vivo. We also investigated a possible relationship between cis-dimer formation and the cell adhesive state in vivo. Interestingly, in several human tumor cell lines that express E-cadherin normally but nevertheless exhibit a decreased adhesive function, we were unable to detect the cis-dimer by our crosslinking assay. Taken together, these data suggest that the cis-dimer of E-cadherin is a fundamental unit at the cell–cell adhesive interface in vivo.

Frequent Loss of α Catenin Expression in Scirrhous Carcinomas with Scattered Cell Growth

To investigate the mechanisms of disruption of cell‐cell contact in scirrhous carcinoma cells, the expression of both E‐cadherin and α catenin, which is an intracellular cadherin‐binding molecule, were determined in scirrhous‐type adenocarcinomas of the stomach and breast using immunohistochemical and immunoblotting techniques. The losses of E‐cadherin expression in gastric and breast scirrhous adenocarcinomas were 18.1% and 0%, respectively, and those of α catenin expression were 54.6% and 75%, respectively. Frequent loss of α catenin expression occurred in scirrhous carcinomas with scattered cell growth in the stomach and the breast and showed no organ specificity. In addition, all the infiltrating lobular carcinomas, which also infiltrate the stroma as single cells, showed no E‐cadherin or α catenin expression. These findings suggest that down‐regulation of either α catenin or E‐cadherin plays a critical role in the disruption of cell adhesion in carcinomas with scattered cell growth.

Cadherin subclasses: differential expression and their roles in neural morphogenesis.

Cadherins homophilically bind cells. Thus, cells expressing identical cadherins adhere selectively to each other, and they do not randomly intermix with the cells expressing other types of cadherins in vitro. Neural tissues express multiple types of cadherins, and the expression of each cadherin type is spatiotemporally regulated within a tissue during development. This molecular family therefore could operate for the sorting of different cell types in the nervous system. The regulation of N-cadherin expression is also important for the early development of the neural tube. The ectopic expression of N-cadherin in Xenopus embryos, which was induced by mRNA injection, led to the disorganization of neural tube structures or the fusion of the neural tube to the epidermis. These results suggest that the precise regulation of cadherin expression at the quantitative as well as at the qualitative level si crucial for neural morphogenesis.

Identification of a gene family of cadherin cell adhesion molecules

Cadherins are a group of functionally related glycoproteins responsible for the Ca2+-dependent cell-cell adhesion mechanism. They are divided into subclasses, such as E-, P- and N-cadherin, which are distinct in immunological specificities and tissue distribution. Cell aggregation experiments suggest that these molecules have subclass specificities in cell-cell binding and are involved in selective cell adhesions. Analysis of amino acid sequences deduced from the nucleotide sequences of cDNAs encoding cadherins demonstrated that they are integral membrane proteins and share common sequences throughout their entire length; average similarity in the sequences among them is in a range of 50-60%. This result provided evidence that cadherins constitute a gene family which encodes adhesion molecules with different specificities. We also showed that, when cells with little cadherin activity were transfected with cadherin cDNAs, they acquired the cadherin-mediated adhesion properties.

The Loss of the Expression of α Catenin, the 102 kD Cadherin Associated Protein, in Central Nervous Tissues during Development

A monoclonal antibody specific for α catenin, the 102kD cadherin‐associated protein, has been characterized and used to describe the expression and distribution pattern of α catenin in adult mice and mouse embryos. This monoclonal antibody recognized an epitope in the middle part of the α catenin molecule of various vertebrate species, and bound to neither vinculin nor αN catenin, which are cytoskeletal proteins with sequence similarity to α catenin. At the early mouse embryo stage (neurulae stage) α catenin was expressed and concentrated at cell‐to‐cell contact sites together with various types of cadherins in all tissues. In embryos at 12.5 days of gestation, the α catenin expression was gradually diminished selectively in central nervous tissues such as brain and spinal cord, and in most of the adult central nervous tissues the α catenin expression was hardly detected. In adult non‐nervous tissues most of the cells examined expressed α catenin. Especially in well‐polarized tissues such as epithelial cells, α catenin appeared to be highly concentrated at cell‐to‐cell adherens junctions where cadherins act as adhesion molecules.

α-Catenin-Deficient F9 Cells Differentiate into Signet Ring Cells

It has been demonstrated that alpha-catenin is frequently lost in diffuse type adenocarcinomas. We have isolated alpha-catenin-deficient mouse teratocarcinoma F9 cells by gene targeting. Wild-type F9 cell aggregates cultured in the presence of retinoic acid differentiated into embryoid bodies with an outer layer of epithelial cells. In contrast, cell aggregates of alpha-catenin-deficient cells did not develop outer layers under the same conditions. The outer surface cells of alpha-catenin-deficient cell aggregates, however, differentiated into epithelial cells as determined by their expression of epithelial marker proteins. These differentiated cells scattered from aggregates and showed signet ring cell morphology, which is frequently observed in diffuse type adenocarcinomas. We have provided clear evidence that a single mutation in the alpha-catenin gene may be a direct cause not only of the scattered properties of cells but also of signet ring cell formation in diffuse type adenocarcinoma.