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The Search for a Nobel Prize-Winning Synapse Machine


2013′s Nobel prize in Physiology or Medicine honors three researchers in particular – but what it really honors is thirty-plus years of work not only from them, but also from their labs, their graduate students and their collaborators.

Winners James Rothman, Randy Schekman and Thomas S?dhof all helped assemble our current picture of the cellular machinery that enables neurotransmitter chemicals to travel from one nerve cell to the next. And as all three of these researchers agree, that process of understanding didn’t catalyze until the right lines of research, powered by the right tools, happened to converge at the right time.

Long before that convergence, though, these three scientists began by seeking the answers to three different questions – none of which seemed to have anything to do with the others.

The Search for SNARE

When James Rothman started out as a researcher at Harvard in 1978, his goal was to find out exactly how vesicle transmission worked.

Vesicles – Latin for “little vessels” – are the microscopic capsules that carry neurotransmitter molecules like serotonin and dopamine from one brain cell to another. By the late 1960s, the old-guard biochemist George Palade, along with other researchers, had already deduced that synaptic vesicles are necessary for neurotransmission – but the questions of which proteins guided these tiny vessels on their journey, and how they docked with receiving neurons, remained mysterious.

Yale University's James Rothman set out to break down the process of vesicle transmission, chemical-by-chemical, reaction-by-reaction. Courtesy of Yale University.

In other words, although researchers had established the existence of this vesicle transmission process, no one knew exactly what made it work, or how.

Rothman, however, had a promising idea – and he had a team of skilled graduate students, including the future biochem professors Thomas S?llner and Paul Kempf, to put his idea to the test. That idea was to try to break the vesicle transmission process down, chemical-by-chemical.

The idea was ambitious, no question – in fact, most researchers at the time thought it was a hell of a long shot – but still, it wasn’t without precedent. Biochemists in the early twentieth century had used this approach to figure out how cells digest sugar. Those earlier researchers had broken sugar metabolism down into steps, broken each step down into chemical reactions, and broken each reaction down into individual chemicals, until they could describe every detail of the process from the molecules up.

As far as Rothman could see, there was no reason this method couldn’t work for the more complex process of vesicle transmission – it just might take more time. How much time it’d take, exactly, was anybody’s guess.

To give his team some extra motivation, Rothman preemptively named a significant complex of proteins they hadn’t discovered yet, but expected to: The complex that hooked incoming synaptic vesicles onto the surfaces of receiving neurons. They named this elusive creature the “SNARE” complex. The first person to describe the chemical composition and behavior of SNARE, Rothman knew, would be describing most of the process by which vesicles of neurotransmitters reach their destinations – and would thus become a scientific legend.

As they began their search for SNARE, Rothman and his team isolated synaptic vesicles from living human neurons, and set about trying to reproduce the vesicle transmission process artificially. Within just a few years, the team publicly announced the descriptions of three previously unknown proteins – NSF, ATPase and SNAP – that they knew must play major roles in helping SNARE do its job. Two other proteins, known as syntaxin and synaptobrevin, also seemed central to SNARE’s abilities.

But even in light of these discoveries, several of the SNARE’s complex’s most crucial components – particularly a protein Rothman had pre-named VAMP, which he knew SNARE needed in order to guide vesicles onto the surfaces of receiving neurons – remained frustratingly elusive as the years went by. Rothman’s team had found plenty of suggestive hints of VAMP; gaps in SNARE’s chemical composition and behavior where something had to fit; but they couldn’t pin down anything definitive.

And so, for years, Rothman’s long-shot quest for the complete SNARE complex continued in vain.

The Yeast Code

At first glance, yeast growth might not exactly sound like a game-changing research topic – let alone one that’d turn out to have anything to do with synaptic transmission in the human brain. But in the late 1970s, the growth patterns of these industrious little cells were whispering hints of big secrets to Randy Schekman.

“When I started my research at Berkeley in 1976,” Schekman says, “my ultimate goal was to figure out how to set up the chemical reactions that power yeast growth in a cell-free environment” – to engineer, in other words, an artificial yeast-growth system that didn’t require any actual yeast cells at all.

Randy Schekman of the Howard Hughes Medical Institute began by studying yeast genetics, and soon started finding yeast genes that coded for proteins also involved in human synaptic transmission. Courtesy of Howard Hughes Medical Institute.

If Schekman and his team could create this chemical system from scratch, he reasoned, they’d be able to do a lot more than just grow yeasts – they’d be well on their way to understanding some of life’s most fundamental biochemical processes: Those that tell living cells when to grow, how long to keep growing, when to reproduce and when to die.

But Schekman knew that creating such a cell-free system wouldn’t be simple. In order to isolate each chemical reaction involved in the process of yeast growth – and, eventually, test whether their cell-free reactions were working as planned – his team would first have to identify the yeast genes that coded for each chemical in that process.

Schekman and his team of grad students – including the future Yale professor Peter Novick – began by engineering a colony of yeast mutants that only grew under very specific temperatures. Since the yeasts’ mutation altered the way they secreted certain growth chemicals, Schekman named them “SEC mutants” – short for “secretion mutants.” By cloning the genes of these SEC mutants and comparing them to the genes of other yeast strains, Schekman’s team started to crack the genetic code that tells yeast how, when and how much to grow.

They made steady progress throughout the 1980s, isolating a long list of yeast genes that coded for proteins involved in the growth process. And it wasn’t long before they noticed something curious about these genes: At least two of them coded for proteins – NSF and SNAP – that Rothman and his team had recently discovered in human vesicles.

And so, by the late ’80s, Rothman and Schekman – the synaptic biochemist and the yeast expert – had started up a steady correspondence that was quickly blooming into a productive friendship. They talked of other proteins that might be involved in SNARE’s behavior, such as synaptotagmin and synaptobrevin, along with an intriguing set of SM proteins that Schekman’s lab had recently sequenced.

Yet between the lines of the reams of data they exchanged, an underlying question always lurked: Might the SNARE complex itself turn up in some equally unexpected place?

The Neuron Protocol

Thomas S?dhof’s research career began in 1986, a full decade after Schekman and Rothman began theirs – but S?dhof wasn’t slow to catch up. He was trained as a neuroscientist, not a biochemist; and he got hooked on vesicle research not out of a love for membranes or cell growth, but out of a longing to understand how the brain moves information from one place to the next.

Stanford University's Thomas S?dhof started out studying the physiology of neurons - and ended up helping discover many components of the cellular machinery that Rothman and Schekman were mapping. Courtesy of S. Fisch.

“I was intrigued by the question of how neurotransmitter messages pass from one neuron to another,” S?dhof says, “so my first postgraduate research project was to try to clone and purify synaptic vesicle proteins.” If he could isolate these proteins – including those whose descriptions Rothman was just then beginning to publish, and whose genetic coding Schekman was starting to unravel – S?dhof thought he might be able to describe the precise process by which neurotransmitter chemicals pass from one neuron to the next: The transmission protocol of the human brain.

S?dhof and his team soon isolated and cloned Rothman’s SNAP proteins, as well as several other synaptic enzymes that Rothman and Schekman had been working on over the past few years.

And then he began to discover proteins they didn’t know about – some of which looked an awful lot like the proteins on Rothman’s “most wanted” list.

The Pieces Come Together

In February 1993, Rothman was on the Harvard campus, as usual, preparing his notes for a lecture he was about to deliver – when a call from his lab came through. It was grad student Thomas S?llner, and it sounded urgent.

S?llner read out the gene sequence of a new protein to Rothman over the phone – and it was clear to both of them, that that protein was VAMP. A chain of other logical realizations followed in a synaptic flash: VAMP was in the vesicle; syntaxin was in the plasma membrane; these two proteins formed a complex together; and that protein complex was SNARE.

After almost three decades of searching, they’d finally found their missing piece.

Even so, the lab’s discovery was preliminary. The researchers still had to prove definitively that this new protein was actually VAMP; that it was the final component of the SNARE complex; that it played the role they thought it did in guiding vesicles to neuron membranes. So as soon as Rothman got back to the lab, he and his team dove in as hard as they could.

Grad student Paul Kempf put in 18-hour shifts at the sequencing machine, night after night. S?llner, who’d first sequenced the VAMP protein, performed test after test on its chemical behavior. And through it all, Rothman crunched the numbers and interpreted the results.

Other grad students couldn’t help noticing the sudden flurry of activity, and poked their noses into Rothman’s office to see what all the excitement was about. As soon as they understood, they too jumped in to offer whatever contributions they could. Within a few days, the project had taken over the whole lab.

SNARE Grows Up

On February 25, 1993 – a mere two weeks after Thomas S?llner’s fateful phone call announcing the discovery of the VAMP protein – Rothman and his team submitted their work for peer review at the prestigious journal Nature. Two weeks later, on March 9th, Nature’s editors wrote back, thrilled by the discovery and clearly recognizing its importance.

But even with its featured position in one of the world’s foremost scientific journals, the SNARE complex didn’t become a celebrity overnight. Throughout the 1990s, other researchers responded with corrections and reinterpretations of the model Rothman had put forth.

For example, Rothman’s original hypothesis claimed that the protein known as NSF helps SNARE hook vesicles to receiving neurons’ membranes – but Dartmouth University’s Bill Wickner chemically isolated NSF’s function in the process, and demonstrated that NSF actually helps break down SNARE complexes after they’ve already played their parts.

As X-ray techniques improved in the late ’90s, Reinhard Jahn and Axel Brunger at Germany’s Max Planck Institute imaged the crystal structure of the SNARE complex, and showed that it was a bundle of four helices – one half of a structure that allows vesicles to literally zipper into place on the membranes of receiving neurons. And as it turned out, the other half of that zipper was the SM protein complex, which Schekman had discovered and sequenced way back in the ’80s.

In the cellular machinery that Rothman, Schekman and S?dhof all helped reconstruct, a SNARE complex - made of synaptobrevin, syntaxin and SNAP-25 - zips together to bind a synaptic vesicle to the surface of a receiving neuron. Courtesy of Danko Dimchev Georgiev, M.D. via Wikimedia Commons.

One issue that no one seemed to be able to resolve, though, was how SNARE complexes could act quickly enough to do their job. Neurons transmit thousands of vesicles in a matter of milliseconds, and SNARE’s unzipping process seemed far too slow for this. And here, Thomas S?dhof reenters the picture. As he continued his work on synaptic vesicles, S?dhof discovered a group of proteins that freeze the SNARE complex less than a millisecond away from completion, so that it’s ready to snap into place and finish its work at an instant’s notice.

A few years later, S?dhof’s lab also discovered how vesicles fuse to the membranes of receiving neurons: The calcium in a protein called synaptotagmin binds to SNARE complexes, releasing the SNAREs from the hold of another protein called complexin, and letting them lock down and release the neurotransmitters they carry.

The SNARE model’s level of detail kept increasing every year – and every new revelation seemed to point back to the same points of origin: The labs of Rothman, Schekman and S?dhof, and the work they’d begun so many years ago.

The Ultimate Prize

As new SNARE revelations kept materializing throughout the late ’90s and early 2000s, Rothman, Schekman and S?dhof all began to garner more and more awards from the biochemistry and neuroscience communities. The bigger and more prestigious those awards grew, the harder and harder it got to avoid wondering when the biggest call of all would come through, and one or more of the researchers would find out they’d won a Nobel prize.

“Until it actually happens, there’s just no way to know,” Schekman says. “Plenty of scientists do spectacular, world-changing work and never win a Nobel in their entire lives. All you can know is that you’ve already won a constellation of other awards, and that the probability of getting that call seems to be going up. But that’s just an inkling, really – and an inkling doesn’t mean much of anything.”

But that call did finally come through, and the three researchers gathered in Stockholm to receive recognition for their three decades of collaboration.

In fact, all three winners agree, one principle whose importance this three-way prize reinforces is that a discovery like this can’t be made solely by geneticists, biochemists or neuroscientists – it takes collaboration and sharing of information across disciplines that might not seem to have much to do with each other.

“Scientific fields tend to be so compartmentalized, even today,” S?dhof says. “In our fields, we all form our little cliques, and we forget that current problems like neurodegeneration, for example; or synaptic plasticity, would really benefit from teams of cell biologists, biochemists, geneticists and neuroscientists all sharing data and collaborating on research like we did.”

And as the latest research begins to integrate older neuro-mapping tools like CT scans and fMRI machines into Big Data-driven efforts like the Human Connectome Project, the molecular level of detail that first got Rothman, Schekman and S?dhof interested in synaptic vesicles looks more important than ever.

Plenty of major neuroscience mysteries remain to be solved – from memory preservation and anti-aging to the nature of consciousness itself – and a strictly classical neuroscience approach is likely to provide incomplete answers. If the example set by these three Nobel winners is any indication, problems like these will only yield their solutions when we venture out of our cliques and start speaking each others’ languages.

The views expressed are those of the author and are not necessarily those of Scientific American.

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