When scientists move from one lab to another, the inevitable change in reagents or mysterious new water properties can vex even the most robust of experimental set-ups. But for Masatoshi Takeichi, a change in the recipe for a trypsin solution when he moved from Kyoto University to the Carnegie Institution Department of Embryology eventually led to his seminal discovery of the cadherin family of adhesion molecules.
Takeichi started investigating the role for divalent cations in cell adhesion as a graduate student in Kyoto, where trypsin treatment of cells disrupted adhesion temporarily before cells reaggregated. When he moved to Carnegie for a fellowship, trypsin treatment led to permanent disruption of adhesion. It turned out that the Carnegie trypsin contained the calcium-trapping molecule, EDTA, as well as the trypsin enzyme. This combination gave Takeichi a clear picture of how to test for calcium-dependent adhesion.
He disrupted Chinese hamster V79 cells with EDTA alone, trypsin plus calcium, or trypsin plus EDTA and assayed for aggregation afterward. The results revealed calcium-independent and calcium-dependent adhesion pathways. More importantly, the calcium-dependent mechanism could be protected from trypsinization by calcium (Takeichi, 1977).
“It was so clear, then, that there was a very important calcium-dependent adhesion molecule,” says Takeichi. “Therefore, I just thought I needed to identify that molecule.” In a preliminary experiment in this study, he did identify a 150-KDa cell surface protein that disappeared from trypsin plus EDTA–treated cells. He remembers at least one reviewer's irritation at the premature correlation.
It would, however, be five more years (and a new cell system) before Takeichi could nail down the identity of the first cadherin molecule (Yoshida and Takeichi, 1982). He recalls several roadblocks while trying to develop an antibody from the V79 cells that could disrupt calcium-dependent adhesion and be used to purify the protein. Then he happened to read a paper by Rolf Kemler using anti-serum made from whole F9 teratocarcinoma mouse cells (Kemler et al., 1977). 8-cell embryos treated with the anti-serum decompacted because the cells rounded up in a manner that was very similar to the behavior of Takeichi's trypsin plus EDTA–treated cells. Once he switched to the F9 cells, Takeichi identified E-cadherin as the first member of a protein family now known to regulate adhesion and, consequently, morphogenesis and tissue structure.
“Masatoshi is remarkable in his ability to use incomplete information in a complex problem to reach the right conclusion. Obviously, he has a very good feel for the biology involved,” says Urs Rutishauser, who had identified another important adhesion molecule, NCAM, the year before (Rutishauser et al., 1976). (NCAM was soon found to be important in adhesion [Rutishauser et al., 1978a] and fasciculation [Rutishauser et al., 1978b] of chick neuronal cells.) He notes that although some peers might have worried about the “purely correlative information” that linked the 150-KDa protein to calcium-dependent adhesion, “nevertheless, he was correct.”
Rutishauser, now at Memorial Sloan-Kettering Cancer Center (New York, NY), says a key component of the 1977 Takeichi study was the observation that calcium-dependent adhesion resulted in more than cells just sticking to each other—there were morphological changes in the adhering cells and their neighbors. The cell spreading led Takeichi to predict, accurately, that calcium-dependent adhesion might regulate morphogenetic behavior of cells. In later studies he showed that cadherins adhered homophilically (Hirano et al., 1987), which he confirmed by transfection (Hatta et al., 1988), and were subject to phosphoregulation (Matsuyoshi et al., 1992).