恶人谷

Signalling lessons from death receptors: the importance of cleavage
By Vishva M. Dixit
The place was Ann Arbor, University of Michigan, in the spring of 1994. Three years earlier, I had switched from studying extracellular matrix proteins to investigating how death receptors induced apoptosis. At the time, only two death receptors were known: tumour necrosis factor receptor 1 (TNFR1) and Fas/CD95. It was a risky move as all my grant support was for research on matrix biology, but it felt like I was just dotting the i's and crossing the t's in this field. I wanted a larger canvas to paint on, and the opportunity to make my mark in an emerging field. Despite knowing that I needed a bigger challenge, I was filled with insecurity and uncertain whether the gamble would pay off. The dire consequences of failure were never far from my mind, especially as an untenured assistant professor. I did not want to be the one demonstrating no progress to the funding agencies, having frittered away their precious resources on some harebrained idea!

So, since I was hell-bent on changing fields, what was I going to work on? Cell cycle? Exciting, but way too crowded. How about the opposite process? Cell death. Now that was intriguing to a pathologist with a morbid streak! In the early nineties, little was known. The morphological description of apoptosis and the beginnings of a pathway were being worked out in the worm Caenorhabditis elegans. I had never seen these worms, so that was not the route for me. What I needed was a cell culture system that was amenable to the induction of apoptosis. Various noxious chemicals caused cells to undergo apoptotic demise, but there was no information on their targets. Perusing the literature, I was captured by observations that one could induce apoptosis in certain cultured cells either with TNF, the cognate ligand for the TNFR, or with an agonist antibody to the Fas receptor. Hurriedly, I wrote to Genentech to obtain recombinant TNF and they supplied me with a veritable ton of material. Dr Yonehara in Japan also kindly provided the agonist anti-Fas antibody.

The first experiments with these 'death ligands' and MCF7 cultured cells were amazing. Initially, the cells did just fine, but after a few hours they underwent a catastrophic 'dance of death'. There was violent blebbing of the cell membrane, dramatic condensation of the nucleus, and the cytoplasm became vacuolated as if on a high boil. Wow! Now here was a pathway worthy of study. What were the death-inducing components engaged by death receptors? There was a body of literature implicating kinases, ion channels, phospholipases, and phosphatases, but I favoured something new. The contribution of the known suspects seemed partial, as if they were peripheral to the central process. So what could it be? An MD/PhD graduate student in the laboratory, Muneesh Tewari, was unleashed on the project. Bright, ambitious, and with a desire to accomplish something important, he tried everything under the sun to inhibit the pathway. Overexpression of cDNA libraries and application of kinase, phosphatase, and lipase inhibitors all yielded nothing. The inhibition, when observed, was always partial.


We were also running out of time. Muneesh needed to graduate and I needed to renew my grants. Something had to give or we were going to be in a whole heap of trouble. Fortunately, a vital clue emerged from the world of C. elegans. The worm death gene, ced3, was discovered to encode a protein homologous to a mammalian cysteine protease called ICE (interleukin-1 converting enzyme). Could ICE or an ICE-like protease operate in the death receptor pathway? I realized this notion could be tested because there was a cowpox virus inhibitor of ICE called CrmA. All we needed to do was transfect CrmA into MCF7 cells and see if we conferred resistance to TNF or the agonist anti-Fas antibody. A quick note to David Pickup at Duke University, North Carolina, garnered us the CrmA expression construct. I was working late, probably on a grant, when Muneesh came running into my office. His face was filled with incredulity, as though he had just unearthed the most astonishing treasure. He pulled me to the microscope and showed me vector-transfected MCF7 cells and CrmA-expressing cells, both exposed to TNF. The vector-transfected cells were dead as door nails but, lo and behold, the CrmA-expressing cells looked the image of health. They were totally oblivious to the added TNF. Indeed, they were proliferating, indicating that CrmA had totally eliminated the death signal. The inescapable conclusion was that a component of the death receptor pathway was ICE or a related protease.

In short order, work from a number of laboratories, including our own, defined a number of mammalian ICE-like proteases, renamed caspases, which were able to induce apoptosis. The question that remained was, how do death receptors activate caspases? At that time it was thought that receptors either served as ion channels or altered phosphorylation and dephosphorylation events. Death receptors, it was assumed, must signal in a similar fashion. Here came the second major surprise: postdoctoral fellow Marta Muzio and graduate student Arul Chinnaiyan were able to show conclusively that death domain-containing receptors signalled through an entirely new mechanism, by generating a protease as the 'second messenger'! Processing of downstream caspase zymogens caused precipitous cleavage of cellular substrates and a rapid apoptotic demise of the cell. The rest is history.

Accidental encounters: the chance to solve a mystery
By Masatoshi Takeichi

IntroductionCells organize into tissues by adhering to one another. Such intercellular associations can be disrupted artificially and, under the right culture conditions, the dissociated cells can re-aggregate and reconstitute their original tissue-like structures, as demonstrated by early pioneering studies. When I entered graduate school, the molecular mechanisms governing these striking cellular behaviours, including the formation of the initial cell–cell contacts, were largely unresolved.

At the beginning of my career, I was interested in lens cell differentiation. Lens epithelial cells differentiate into lens fibres, a process that was thought to depend on unidentified factors released from the retina. I set out to characterize these putative factors by culturing retinal cells and collecting the culture medium 'conditioned' by these cells, thinking that it might contain the factors I sought. But when I grew lens cells in this conditioned medium, nothing seemed to happen. After much fruitless effort, I finally noted a difference; lens cells suspended in the conditioned medium attached to the culture dish more slowly than lens cells in the control medium. This unexpected effect had nothing to do with lens differentiation, but attracted my interest nonetheless, for I felt it should be somehow possible to analyse the underlying mechanisms. But the tools needed to take a mechanistic approach to problems in cell differentiation had yet to be developed, and I eventually gave up on the lens.

I was in Tokindo Okada's laboratory at Kyoto University at this time. Although his main interest was in cell differentiation, he encouraged students to learn about morphogenesis as well. He inspired his students to gain broad insights into developmental mechanisms, and provided us with a learning environment that, I believe, was critical in developing my interest in topics such as cell adhesion, which were not widely popular among developmental biologists.

I continued studying cell adhesion and subsequently found that the mechanisms of cell–cell and cell–substrate adhesion require different divalent cations (Ca2+ and Mg2+, respectively) and through this work I became convinced that cells must have multiple adhesion mechanisms. But as the necessary techniques remained unavailable, I still could not test this idea at the molecular level. Around this time, I went to do a postdoctoral fellowship in Richard Pagano's laboratory at the Carnegie Institution of Washington, Department of Embryology, and began to explore the mechanisms behind liposome–cell membrane interactions. Soon after the move, however, I noticed something strange. I generally used trypsin to dissociate cells, which would normally re-aggregate when cultured in suspension. But when I used the Carnegie recipe do the same thing, the trypsinized cells never re-aggregated. This surprised and interested me, and I set out to solve the mystery. It turned out that the Carnegie trypsin solution contained EDTA to remove divalent cations, whereas my usual solution did not. I confirmed that the presence or absence of Ca2+ in the trypsin solution was the key to the difference I had observed. This led me to hypothesize that cells have an adhesion mechanism that can be disrupted with trypsin, and that Ca2+ confers a protective effect against trypsin digestion. Since the re-aggregation of the cells equipped with this hypothetical mechanism also required Ca2+, I called it the Ca2+-dependent adhesion system. I had a strong feeling that this mechanism must be crucial for cell–cell adhesion in animal cells, as it had previously been suggested that Ca2+ is indispensable for the maintenance of animal tissues, and so I decided to follow up on this finding.


Identifying the molecular mechanism underlying Ca2+-dependent adhesion, however, was not an easy task. One promising approach was the immunological one, which was introduced by Günther Gerisch's group to identify adhesion molecules of the cellular slime moulds. The idea behind this approach was that if I could raise antibodies that are able to block cell–cell adhesion, it would enable me to identify antibody targets, which would presumably be adhesion molecules. I tried injecting rabbits with cells, which I had used in the experiments at Carnegie, but these never led to the production of the blocking antibodies I was after. One day, however, I came across a paper by Rolf Kemler and colleagues reporting that antibodies raised against teratocarcinoma cells blocked the compaction of early mouse embryos. Given the morphological similarity between embryonic compaction and Ca2+-dependent cell aggregation, I suspected the underlying mechanisms might be related as well. Indeed, when I switched to teratocarcinoma lines, I was finally able to obtain blocking antibodies. It was these antibodies that led us to identify the first of a large family of molecules now known as cadherins.

Looking back on my early research, it is clear that the struggle to account for some unexpected finding or other has often brought me to a turning point. As scientists, we need to keep ourselves attuned to the uncommon and to avoid blinkering ourselves with dogma. Admittedly, these days I tend to propose rationally designed experiments to my postdocs and students, but I always strive not to overlook any unexpected results from their experiments, and to emphasize to them the importance of this attitude for the advancement of science.