KEYSTONE MILLENNIUM MEETING: CONFERENCE REPORT September 2000 © 2000 Elsevier Science Ltd. All rights reserved 7

The cell

Know your destinyHow do primordial stem cells begin the transition to themore specialized cells that form our tissues and organsduring fetal development? This is a question raised byTony Pawson, a cell biologist at the Samuel LunenfeldResearch Institute of the Mount Sinai Hospital in Toronto.Pawson believes that the microscopic circuitry controllingthe inner workings of our cells holds the secret to howour bodies work as a whole, and how diseases result fromfaults in the wiring.

Pawson and his team are focusing on proteins calledreceptor tyrosine kinases (RTKs) that straddle the cellmembrane. Essentially, these are the information gatewaysthat allow our cells to pick up signals from outside andturn them into instructions that tell the cell to do some-thing different. “The receptor crosses the outer membraneof the cell and acts as an antenna,” says Pawson.“On the out-side, it binds very strongly to a signalling molecule, such asinsulin,” he says. “On the inside, it has portions to com-municate with other proteins inside the cell.” ActivatedRTKs might, for example, trigger proteins that feed intothe cell nucleus to activate new genes. Some trigger cellenzymes into action and others commission a spot ofmaintenance to keep the cell in good working order.

The big breakthrough came just over a decade ago whenPawson discovered a key link in the signaling chain called theSH2 domain.They found that many proteins, from enzymesto transcription factors, have an SH2 domain, which enablesall of them to relay information from activated RTKs.

SH2 domains were just the start, however. Pawson andothers have since discovered many more domains, each onewith its own specialized function in the cell’s circuitry.Some have obvious parallels with electrical circuits.Adapterproteins, for example, do exactly the same job as plugadapters, connecting proteins that couldn’t otherwise linkup. Some domains are more sinister. Death domains, astheir name suggests, order cells to die.

Pawson’s latest work involves a signal that kick-starts thetransition of raw, primordial stem cells into more specializedcells during embryonic development and tissue mainte-nance. The division of a stem cell produces two unequaldaughters. One is still a stem cell, but the other has begunto differentiate into a specialized type of cell. The teamtraced the signal to a protein domain called PTB, whichwas, in this case, located within a modular protein knownto be active in one daughter cell but not the other. Pawson

says that clues to how cells can be switched from one typeto another could be vital in newly emerging fields such astherapeutic cloning, which holds the prospect of makingorgans for transplant from a patient’s own tissue.

Andy Coghlan

Variation: virtue or vice?An old genetic trick could allow biologists to explore whatLeland Hartwell says is emerging as an important evolu-tionary property of organisms – the ability to accumulatea huge amount of genetic variation.

Natural populations, including humans, contain a largeamount of genetic variation. This includes obvious differ-ences, such as height and skin colour, as well as hidden ones.The ability of human cells to repair DNA damage varies byas much as 100-fold between individuals.What’s remarkable,says Hartwell, a geneticist at the Fred Hutchinson CancerResearch Center in Seattle, is that such tremendous variationisn’t harmful. On the one hand, genetic variation helpspopulations survive in changing environmental conditions.On the other, when conditions are constant, many of thesegene variants might be detrimental if fully expressed.

Hartwell and his colleague Stanislas Leibler of PrincetonUniversity, think this suggests that cells contain ‘biologicalcircuits’ of molecules that buffer the potentially harmfuleffects of genetic variation. So when one part of a circuitchanges by mutation, another part can balance the effect –a property that Leibler dubbed robustness. “This seemslike an important evolutionary principle,” says Hartwell.“It permits the accumulation of variation by limiting itsimpact on expression.”

Hartwell has proposed a way to find these robust cir-cuits. He says that by turning a familiar genetic phenomenonon its head, researchers can start attacking this problem.Certain pairs of mutated genes are individually innocuousbut kill the cell in combination. Geneticists frequently searchfor such combinations of so-called synthetic lethal mutationswhen hunting for proteins that interact with each other.

But Hartwell realised that synthetic lethal combinationsare a form of robustness. If mutations in gene A don’tbecome apparent until gene B is mutated, and vice versa,each is essentially buffering the defect in the other. His lab recently discovered an example of this relationshipwhen they searched for synthetic lethal genes that could

…microscopic

circuitry

controlling the

inner workings of

our cells holds

the secret to how

our bodies work

as a whole, and

how diseases

result from faults

in the wiring…

Life in the fast laneRecent discoveries have greatly improved our ability to navigate around the cell. The human genome sequence should increase the number of signposts to guide us.

KEYSTONE MILLENNIUM MEETING: CONFERENCE REPORT September 2000 © 2000 Elsevier Science Ltd. All rights reserved8

The cell

compensate for a the deletion of the yeast gene MEC1,which encodes a protein that helps halt chromosomereplication in response to DNA damage.The deletion isn’tdeadly by itself.

Most of the genes they found were involved inmetabolizing nucleotides, the building blocks of DNA.Hartwell believes these genes might form a circuit thatbalances interruptions in DNA replication with the avail-ability of nucleotides.

Hartwell thinks that some general ideas about robust-ness can now be gleaned from the vast literature on syn-thetic lethal mutations. But because these experimentsweren’t done with gene circuits in mind, many will behard to interpret. “Unfortunately, this is not the kind ofdata we would like in studying this process,” he says.

Philip Cohen

Chop and changeIn 1978, a film made in by Marc Kirschner, a cell biologistnow at Harvard University, showed that frogs’ eggs couldbe seen moving rhythmically through the cell cycle. Thecells bounced up and down once every few hours, aboutas often as they would have divided, had they been able to.The surprising part was that the cells shouldn’t have beenmoving at all.They had no nuclei.

At that time, the nucleus was thought to be the pace-maker of the cell cycle, in the course of which a cell repli-cates its DNA and splits in two. Cells tend to ball up and thencontract in the middle as they prepare to split. Without anucleus, it was thought, these movements would stop, justas a heart without a pacemaker will cease to beat.

Kirschner’s discovery that a cell can keep ‘beating’without a nucleus hardened his determination to learnwhat actually controls the cell cycle. Over the past 20 years,he and many others who have joined the field have devel-oped a comprehensive picture of what controls this activity.With the help of that picture, Kirschner believes, we willeventually be able to control the division of human cellsclosely enough to stop cancer, grow whole organs, andpossibly even replace missing limbs.

When Kirschner was experimenting with frogs’ eggs,the prevailing theory was that the cell cycle began withevents in the nucleus such as DNA replication.The subse-quent stages of mitosis, then followed like falling domi-noes. So biologists around the world were astonished tosee Kirschner’s nucleus-free eggs bouncing up and downin time with the normal cell cycle.

After years of searching, Kirschner and others discov-ered signalling systems that might be connected to the cellcycle. But these didn’t work like the other biochemicalsignals biologists knew about, which usually involve pro-tein factors activating one another in a signalling cascade.Instead, the driving force behind the cell cycle turned outto be destruction.

In the early 1980s, protein destruction, or proteolysis,was thought of as nothing more than a form of recycling. Bydismantling the cell’s used or damaged proteins, proteolysisinsured a continuous supply of building blocks for new pro-teins. “This field was sort of sitting on the sidelines,” saysKirschner. “No one was paying much attention to it, becausesynthesis was much more interesting than degradation.”

As it turns out, proteins called cyclins build up gradu-ally in the cell until they reach a critical concentration, atwhich point they set off a series of events leading to mitosis.But Kirschner’s team discovered in 1989 that before thecell can finish mitosis it must chop up its cyclins, resettingthe cell for another round.

Many old proteins in the cell are recycled in membrane-bound bags of proteolytic enzymes called lysosomes. Butresearchers soon learned that this routine refuse removalsystem isn’t used for cyclins. Instead they are singled outfor a highly selective destruction process. First, tiny pro-teins called ubiquitins latch onto the cyclins, flaggingthem for destruction. Then a massive protein-engulfingmachine, the proteasome, spots the ubiquitin flag andmoves in for the kill. Shaped like a short tube, the protea-some ingests proteins at one end and spits the fragmentsout from the other.

This ubiquitin–proteasome system turned out to play arole in many cellular processes, including DNA repair, thecell’s response to stress and the activation of genes. It is alsoinvolved in abnormal processes such as nerve degenerationand cancer. As a result, proteolysis, a process once almostignored, now occupies a central place in cell biology.

One key question remaining about the cell cycle is howthe cell knows when to ‘ubiquitinate’ the cyclins – or anyof the several other cell cycle proteins it targets – to setproteolysis in motion. In the case of the cell cycle, a clus-ter of proteins called the anaphase-promoting complex(APC), named after the part of mitosis when cyclins aredestroyed, seems to be at the heart of the decision.The APC’sjob is to attach the ubiquitin tag to the target protein.

Kirschner’s group recently reported the discovery of ashort protein sequence common to most known APC tar-gets. He hopes that this sequence will lead to the identifi-cation of other targets of APC, and perhaps shed some lighton the regulation of proteasome degradation. Already,Kirschner says, several researchers have contacted him withnews of possible new regulatory functions for APC thatthey identified with the help of the sequence.

A better understanding of the signals in cell divisioncould lead to ways of correcting cell-cycle malfunctionssuch as cancer, Kirschner says. Doctors would also like tobe able to trick cells into dividing in response to artificialsignals. “I think we will figure out how to get cells to pro-liferate,” Kirschner says. “That will lead to the ability toregenerate nerves, liver, heart muscle and maybe limbs.”Salamanders can regenerate limbs, so nature has proventhat it is possible. “We’ve just got to figure out how toimitate that process,” he adds.

“The ability of

human cells to

repair DNA

damage varies

by as much as

100-fold between

individuals. What’s

remarkable is that

such tremendous

variation isn’t

harmful.”

Leland Hartwell