Shoichiro Tsukita was one of the best, well known, and respected cell biologists in Japan, and, sadly, pancreatic cancer took him away much too early at the age of 52 in 2005. His amazing courage and dedication to science were manifested by the fact that he spent the last few months of his life writing a small book for young scientists on the discovery of claudin. Tsukita's wife and partner in science, Sachiko Tsukita, a brilliant scientist in her own right, helped to translate the book into English. Our thanks go to her and the Japanese publishers of the book, who have allowed the English translation to be made available to ASCB members and other scientist worldwide. Additional thanks are due to the ASCB leadership for providing the incentive and giving permission for this endeavor. Finally, we are grateful to Masatoshi Takeichi and Rockefeller Press for allowing the ASCB to publish a slightly modified version of Takeichi’s memorial note for Tsukita, entitled "Shoichiro Tsukita: a life exploring the molecular architecture of the tight junction." The note was originally published in the Journal of Cell Biology on January 30, 2006.
--Mina J. Bissell
On December 11, 2005, Shoichiro Tsukita died at the young age of 52, after 14 months of treatment for pancreatic cancer. Early in his career, Tsukita succeeded in isolating and purifying the adherens junction with his wife Sachiko Tsukita, an accomplishment that he followed up with an impressive series of discoveries of cell adhesion and cytoskeletal molecules, including what may have been his greatest contribution to the field, the identification of occludin and the claudin family of molecules. These were watershed discoveries in the study of the molecular nature of tight junctions.
The book, Claudins, was written by Shoichiro in the last few months of his life. The collaboration of the two Tsukitas started with the discovery of the desmosomal protein, desmocalmin. In those days, the desmosome was still terra incognita at the molecular level. It was not long, however, before they turned from the desmosome to the adherens junction, a cell–cell adhesion structure that Shoichiro and Sachiko were the first to isolate intact, from preparations of rat liver hepatocytes (Tsukita and Tsukita, 1989). These purified adherens junctions represented something of a mother lode for Tsukita, whose scrutiny of their molecular composition yielded a cornucopia of important discoveries , and indeed launched him down a path that he would pioneer for the rest of us throughout the remainder of his career.
The first molecule that the two Tsukitas teased out of the complexity of the adherens junction (AJ) fraction was radixin, one of the founding members of the eponymous ERM family; Sachiko Tsukita has gone on to advance the study of this molecule in her own work. ?-Catenin was the next molecule to emerge from the AJ trove, a finding that contributed immensely to our understanding of the cadherin cell adhesion machinery. The subsequent discovery of ZO-1, a known tight junction (TJ) protein, in what until then had been thought to be a pure AJ fraction led Tsukita to surmise that the fractions were in fact enriched with TJ components as well. In 1993 with Mikio Furuse, the team developed a monoclonal antibody from their AJ preparation that recognized a protein in the tight junction. They cloned this 4-pass transmembrane protein and christened it occludin (Furuse et al., 1993), a name taken from the TJ’s alternative appellation, zonula occludens, marking the identification of one of the first TJ-specific membrane proteins. Tight junctions are adhesive structures found in the most apical regions of epithelial cell layers, where they prevent the unregulated passage of matter between cells. A number of tight junction cytoplasmic factors had been described relatively early on, but for many years its membrane organization remained unresolved at the molecular level.
After the excitement of the occludin discovery, Tsukita was later to experience disappointment on finding that tight junctions form even in cells in which the occludin gene had been deleted. At the same time, he and Furuse had already decided that there must be another membrane protein at work in the tight junction, a prediction Furuse proved correct by sifting through a subfraction of occludin-positive membrane from the purified adherens junction. His search yielded a pair of novel proteins, which he named claudin-1 and - 2 (Furuse et al., 1998a). As predicted, the claudins were shown to localize at the tight junction, and even more happily, caused fibroblasts (which do not normally form tight junctions) to form characteristic TJ strands when ectopically expressed (Furuse et al., 1998b), a demonstration that provided the conclusive evidence in the long search for an essential TJ membrane protein.
Research into claudins blossomed rapidly into a fertile and active field of molecular biology, and this protein family is now known to include at least 24 members, highlighting a hitherto unsuspected diversity among tight junctions that continues to challenge and reward investigators. The discovery of the claudins has led to a number of new revelations. As just one example, when investigators found that claudin-1 knockout mice exhibited an unusual dry skin phenotype (which was remarkable in that the skin was tradition-ally believed to lack tight junctions), it led to the discovery of a new role for this form of cell adhesion in preventing water loss through the body surface by a TJ-dependent barrier (Furuse et al., 2002). In the blood–brain barrier as well, a claudin (this time, claudin-5) was again shown to be indispensable to physiological function (Nitta et al., 2003). Work by other scientists has shown that genetic defects in other claudin proteins can lead to inherited disorders including kidney diseases (Simon et al., 1999; Müller et al., 2003) and deafness (Wilcox et al., 2001), demonstrating the impact of Tsukita’s work on the biomedical community.
Even during his illness, Tsukita was engaged in discovering an important new molecule, tricellulin (Ikenouchi et al., 2005). Claudins function at tight junction regions that involve only two cells, but it is known that cell layers also include junctions where three cells meet: what are known as tricellular contacts. Just as with their two-cell cousins, these tricellular TJs must form a functional epithelial barrier. During a comprehensive screen of 4-pass transmembrane proteins, Junichi Ikenouchi, a grad student in Tsukita’s lab, uncovered a molecule that localized at tricellular contacts, adding yet another achievement to the list of discoveries by Tsukita’s lab of molecules whose existence was predicted by no more than the fact that “they ought to be there.”
The acuity of Tsukita’s scientific instincts was formidable, and he had an equal gift for observation. Findings from histological studies may seem unexciting at first glance to some, and I suspect that they often meet with a colder welcome from peer reviewers than do molecular biology manuscripts when submitted for publication. But at the end of the day, structures tend to have the final say in determining an experiment’s validity, and I’m certain that Tsukita’s work will live on in perpetuity in textbooks of cell biology. The great majority of his many important primary research articles were published in the Journal of Cell Biology, for many years the official journal of the American Society for Cell Biology.
The ASCB has now decided to post Shoichiro Tsukita’s book on the discovery of the Claudins in English on its website.
-- Masatoshi Takeichi
Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol. 123:1777–1788.
Furuse, M., K. Fujita, T. Hiiragi, K. Fujimoto, and Sh. Tsukita. 1998a. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J. Cell Biol. 141:1539–1550.
Furuse, M., H. Sasaki, K. Fujimoto, and Sh. Tsukita. 1998b. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in ~broblasts. J. Cell Biol. 143:391–401.
Furuse, M., M. Hata, K. Furuse, Y. Yoshida, A. Haratake, Y. Sugitani, T. Noda, A. Kubo, and Sh. Tsukita. 2002. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin- 1-de~cient mice. J. Cell Biol. 156:1099–1111.
Ikenouchi, J., M. Furuse, K. Furuse, H. Sasaki, Sa.Tsukita, and Sh. Tsukita. 2005. Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J. Cell Biol. 171:939–945.
Müller, D., P.J. Kausalya, F. Claverie-Martin, I.C. Meij, P. Eggert, V. Garcia-Nieto, and W. Hunziker. 2003. A novel claudin 16 mutation associated with childhood hypercalciuria abolishes binding to ZO-1 and results in lysosomal mistargeting. Am. J. Hum. Genet. 73: 1293–1301.
Nitta, T., M. Hata, S. Gotoh, Y. Seo, H. Sasaki, N. Hashimoto, M. Furuse, and Sh. Tsukita. 2003. Size-selective loosening of the blood-brain barrier in claudin-5–de~cient mice. J. Cell Biol. 161:653–660.
Simon, D.B., Y. Lu, K.A. Choate, H. Velazquez, E. Al-Sabban, M. Praga, G. Casari, A. Bettinelli, G. Colussi, J. Rodriguez-Soriano, et al. 1999. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science. 285:103–106.
Tsukita, Sh., and Sa. Tsukita. 1989. Isolation of cellto-cell adherens junctions from rat liver. J. Cell Biol. 108:31–41.
Wilcox, E.R., Q.L. Burton, S. Naz, S. Riazuddin, T.N. Smith, B. Ploplis, I. Belyantseva, T. Ben-Yosef, N.A. Liburd, R.J. Morell, et al. 2001. Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell. 104:165–172.