The emergence of integrins: a personal and historical perspective.

I never intended to work on cell adhesion. My involvement in this fascinating field for the past 30 years originated in a desire to find molecular differences between the surfaces of normal and tumor cells. In the early 1970s, viral transformation of cultured cells had been shown to yield tumorigenic cells that differed in their growth properties, in their dependence on serum factors and on attachment to substrates and in their morphology in culture. These transformed cells grew more than their normal progenitors and we did not know why. In 1971, I went to Michael Stoker's laboratory at the Imperial Cancer Research Fund (ICRF) in London as a postdoctoral fellow. He was an expert on transformation by polyoma virus and I planned to investigate early biochemical alterations induced by the virus that might explain the altered cell behavior. It soon became clear that the so-called “abortive transformation” by polyoma virus was not amenable to biochemical analyses so I looked around for something else to do. Many people in Stoker's lab were working on purifying growth factors from serum but that did not appeal to me. On the other hand, these serum factors promoted cell growth, presumably by binding to “receptors” on cell surfaces. There were also indications from studies with carbohydrate-binding lectins that the surfaces of transformed cells differed from those of normal cells in some way (Burger, 1973) although the molecular basis for these differences was completely unclear. In fact, we knew rather little indeed about the molecular structure of the cell surface of any but a few simple cells such as erythrocytes. The fluid-mosaic model proposing proteins floating in a lipid bilayer had just been formulated (Singer and Nicolson, 1972). So, I decided to try and find out what proteins were on the surfaces of normal and transformed cells and whether they differed. My underlying hypothesis was that some alteration in the surface might underlie the difference in growth control. I tried various surface labeling reagents including synthesizing “Bretscher's reagent” from 35S-methionine (Bretscher, 1971), with the side product of liters of radioactive ether and cooling fluid from the paper electrophoresis tanks—that did not seem to be a practical approach, especially since the reagent only worked effectively above pH 8 and that made the cells leaky. Then I tried the 125I-iodide/lactoperoxide/glucose oxidase system developed in work on erythrocytes (Phillips and Morrison, 1971). We had no fume hood so I took seaweed pills to preblock my thyroid each day and labeled dishes of cells on the open bench. The initial results were encouraging—I saw multiple labeled bands on the, then newly invented, SDS slab gels but initially no differences between normal and virally transformed cells. One day early in 1973, I was looking over my results and realized that all the normal cell lanes had a lot of 125I label stuck at the top of the 10% polyacrylamide gels, whereas the transformed cells had much less or none. The next day I ran some lower percentage cells and, “eureka,” there was a major high molecular weight band (~250 kDa) in the normal cell lanes but it was greatly reduced in polyoma-transformed cells and absent in cells transformed by hamster sarcoma virus (now known to harbor H-ras). Control experiments showed that this protein was removed from cells by low doses of trypsin, an additional criterion for cell surface location. This was fibronectin, although at the time I called it LETS protein (large, external transformation-sensitive). Its loss on transformation or proteolysis seemed to correlate with changes in the lectin agglutinability of cells and in their proliferation, known to be stimulated by transformation or by proteolysis (Hynes, 1973). This was all very exciting and, for a while, I iodinated any cell I could get my hands on until I eventually achieved a minor 125I contamination of my thyroid and ICRF built a fume hood so that I could continue. The loss of LETS protein seemed to be a consistent correlate of oncogenic transformation and we initially thought that we had discovered a surface growth regulatory molecule. It soon became clear that it did not behave like a detergent-soluble integral membrane protein (Graham et al., 1975). Antibodies, first in the hands of Antti Vaheri and Erkki Ruoslahti (Wartiovaara et al., 1974), and later in my own lab at MIT (Hynes et al., 1976; Mautner and Hynes, 1977), showed a fibrillar pattern of staining. When we purified the protein and added it back to cells that lacked it, that had little effect on their growth rate but it had a major effect on their adhesion and morphology (Ali et al., 1977) and on cell migration (Ali and Hynes, 1978). Others also reported effects on cell adhesion and motility (Willingham et al., 1977). So, rather than identifying an integral membrane protein involved in growth control, it seemed clear that we had in our hands an extracellular matrix molecule involved in adhesion. Years later it became clear that fibronectin and integrins do, after all, play very important roles in controlling cell proliferation and survival but, in the mid-1970s, our attention switched to cell adhesion, cytoskeletal morphology and cell migration, all of which were affected by LETS protein/fibronectin. How did that lead to integrins?