Sticking it out with tight junctions

f patience is a virtue, then Daniel Goodenough and Bruce Stevenson earned their wings in the pursuit of the first tight junction protein. Stevenson spent seven years as a graduate student with Goodenough (Harvard Medical School, Boston, MA) before the two worked out the conditions to purify fragile tight junction–enriched preparations from mouse liver (Stevenson and Goodenough, 1984). It was enough to jump-start the field of tight junction biochemistry, some 20 years after the first morphological description of these junctions, which give epithe-lial cells their ability to seal off body compartments (Farquhar and Palade, I 1963). The preparations yielded multiple protein bands—two major and six minor. Stevenson took the project with him to Mark Mooseker's lab at Yale University (New Haven, CT) as a postdoc and collaborated with Janet Siliciano in the Goode-nough lab. Using the tight junction fraction, they screened mono-clonal antibodies by looking for localization to the junctions by immunofluores-cence EM. Commuting back up to Boston in his Ford Fiesta, Stevenson and Sili-ciano worked on the tedious process of antibody production. At first, some of their hybridomas would turn up positive, only to be lost upon cloning out to a 96-well plate. On a Saturday before heading out to the beach, Stevenson says he remembers checking two plates, and being prepared to throw them out, only to find all of the 48 wells turning up positive. He cancelled his vacation and set to cloning the colonies out for the rest of the day. His perseverance led to the antibody that identified the first tight junction protein, ZO-1 (Stevenson et al., 1986). The protein occurred in many other epithelial cell tight junctions including those of rat liver and mouse colon, kidney, and testis. The widespread distribution of the protein, cloned a few years later (Anderson et al., 1989), argued that it could be a " ubiquitous component of all mammalian tight junctions. " More than ZO-1 But it was clear early on that, as a peripheral membrane protein, ZO-1 was not the integral junction-forming protein. Finding this protein was still up for grabs, and those in the United States went back to their tight junction preps to try to generate other antibodies. But contamination with the very antigenic ZO-1 and gap junction proteins foiled those efforts. In the end, the prize went to Shoichiro Tsukita's group, then at the National Institute for Physiological Sciences in Okazaki, Japan. " Tsukita, …