Profile on Stan Burgess: Molecules in Motion
Devoted to dynein: Against all odds, Dr Stan Burgess captured the motor protein in motion.
Late last year, Dr Stan Burgess from UK-based Astbury Centre for Molecular Biology, University of Leeds, and colleagues, revealed the very first images of the elusive motor protein, dynein, in action.
For decades, biologists had struggled to understand how the cell's three molecular motors - kinesin, myosin and dynein - move, and thanks to its sheer size and complexity, dynein has baffled most. Not any more.
Using cryo-electron microscopy to capture thousands of images of this mysterious molecule at different stages of motion, the Burgess-led team pieced together short, sharp movies that laid out its structure and, quite literally, its stride.
As the team reported in Nature Communications, the motor protein moves along a microtubule track by swinging two stalk-like 'arms' to alternatively grab hold of binding sites with what they have called a 'grappling hook'.
Dynein dimer 'hanging' from microtuble. [Leeds University]
"Myosin and kinesin had been expressed, engineered, purified and crystallised yet the very large and bulky dynein was so difficult to work with," he explains. "But following so many years of research, we got the final images and I still can't believe it."
Single-molecule images of dynein: background; negative stain, foreground; cryo-EM [Burgess et al]
Burgess's fascination with structure and form started at an early age. At school, the young student was torn between the arts and sciences, but opted for the latter, figuring his passion for drawing - with in his words, a mathematical leaning - could be fulfilled at home. Science couldn't.
Astronaut ambitions followed with Burgess selecting Mathematics, Physics and Chemistry at A'Level, but as university approached he suddenly switched his choice of degree from astrophysics to physiology after reading about insulin and its structure.
"I became completely captivated by biology and figured I could still become an astronaut by working for NASA as a space physiologist," he says.
Towards the end of his physiology degree at Bristol University, UK, Burgess was taught about about cilia and flagella by lecturer Dr David Woolley from the Department of Physiology, sparking his life-long obsession with motor proteins. These whip-like appendages extend from cells to move liquid past the cell surface, and for single cells such as sperm, enable movement.
"Researchers had used a laser to slice a live sperm cell; the tail continued to wriggle which meant the motor was inside the tail," explains Burgess.
"I was shown a deep-etched rotary shadowed electron microscope image of the motor proteins underneath the plasma membrane and you could see very single one of them," he adds. "It was amazing."
Crucially, the image had been captured by US-based Washington University biophysicist, Professor John Heuser, via his groundbreaking quick-freeze, deep-etch rotary-replication electron microscopy method.
Designed to image fleeting events within cells, a specimen would be slammed against a cooled copper block to vitrify its surface, fractured under vacuum with a cryomicrotome, partially freeze-dried and coated with platinum and carbon to form a replica of its exposed, fractured surface. The sample would then be thawed and the replica cleaned, ready for electron microscopy.
"I thought it was astonishing that this could work at all and that such tiny structures could survive such a method," points out Burgess. "Heuser's images were by far the most captivating I had ever seen."
Come 1986 and the end of his degree, Burgess moved to York University of study for an MSc in Biological Computation, as in his words: "I knew computers were going to be important." A year later, and with image processing knowledge in hand, he spotted an advert for a PhD on sperm motility with his former Bristol lecturer, David Woolley, applied and got it.
Here, he was tasked with using Heuser's quick-freeze, deep-etch method to capture different nucleotide states and observe changes in dynein motor proteins, in avian sperm cells. One of only three laboratories, worldwide, following Heuser's method, the young researcher seized an opportunity to visit the pioneer, and learn how to perfect the technique.
"This was a real thrill and Heuser had already described many structures for the first time using his method," he says. "As far as I was concerned he was top of his game in electron microscopy as he could see things that nobody else could see."
At the same time, he drew on his new found passion for image processing, and started to apply Fourier transforms to his images to improve quality. His method worked and come 1991 he had published somewhat controversial images of the outer and inner dynein arms in flagella from the sperm cells.
As he points out, the research community simply didn't believe that long thin stalk-like arms could literally pull the bulky motor protein along its microtubule path.
"So we went to a lot of effort to reconcile the different TEM methods, and using ultra-thin serial sections of axonemes [flagella cores] could see these stalks, providing evidence of these so-called B-links," he says.
But despite success, dynein was an enormously difficult protein to work with and research funds dried up. Burgess left academia, but after several months still wanted to 'solve the axoneme', so with help from former PhD supervisor, Woolley, applied for a grant application with The Wellcome Trust.
In a research first, he proposed using a new computer method, single particle image processing, to determine dynein movement. Trawling library journals, Burgess had noticed the technique had been applied to isolated ribosome molecules, so why not dynein?
Raw cryo-EM image of dynein-microtubules in ATP, from work published in Nature Communications.
The grant came through and Burgess eagerly visited Marin van Heel, one of the developers of the single particle image processing software, to share his thoughts.
"He was giving a seminar in Cambridge, I showed him my replica images and asked if it would be feasible to apply single particle image processing to them," says Burgess. "He said 'no', I was crestfallen."
According to the researcher, his replica images were high contrast, had unusual noise features and the motors were very closely packed so isolating a single molecule wasn't easy.
Still determined as ever, Burgess went home, taught himself how to use 'SPIDER' - System for Processing Image Data from Electron microscopy - and met success.
Come 1995, his first results on dynein arms in motion were published in the Journal of Molecular Biology. Yet still this wasn't enough to secure more dynein research funds, and he found himself out of work again.
However, word had spread that Burgess had left academia, and this time, animal cell biologist Professor John Trinick, then at Bristol University, approached the young researcher to see if he would apply single particle image processing to images of the less complex motor protein, myosin.
"Amid financial hardship, I agreed to a one month contract," says Burgess. "And then another came, and then another, and this went on for a year."
By 1997, Burgess, Trinick and colleagues had published single particle analysis results on myosin, in the Journal of Cell Biology. Soon afterwards Burgess wowed audiences at the 'European Muscle Conference' with short videos of myosin movement, and as he laughs: "Having lost the love of my life, dynein, I was now a committed myosin guy."
In love or not, Burgess's contributions to myosin research were significant, and in 2000, his research on myosin took the cover of Nature with results also being published in myriad journals. But the love affair with dynein was far from over.
While working on myosin, Burgess came across research from a group of Japan-based researchers, led by Professor Kazuhiro Oiwa from the Kansai Advanced Research Center.
As he points out, the researchers had produced negative stain images of purified dynein molecules, that 'looked amazing', but hadn't considered applying image processing.
Collaboration began and as Burgess highlights: "We were looking at detail that nobody had even seen before in dynein."
"So we wrote a paper, sent it to Nature, and it made the cover," he adds. "My key ambition in science, believe it or not, had been to design a cover, and I'd managed to do it twice in three years."
At last, success finally brought sustainability, and Burgess's dynein research continued apace.
Having moved to Leeds University in 1997, and joining forces with biophysicist Professor Peter Knight in 2002, results from applying single particle analysis were coming in thick and fast. What's more, researchers worldwide were getting to grips with the unwieldy motor protein.
Cryo-electron microscopy and single-particle analysis were being more and more widely used to unravel dynein and crucially, researchers had discovered ways to express and engineer dynein.
By now, Burgess, Knight and colleagues were working fastidiously with Professor Kazuo Sutoh from Life Sciences, University of Tokyo, and now at Waseda University, who could label dynein with GFP-based tags.
"We managed to deduce so much about the structure of the dynein motor from negative stain electron microscopy and GFP tagging," highlights Burgess.
"We eventually published in Cell, in 2009, and it was a phenomenal piece of work," he adds. "We'd inserted tags at numerous locations within the motor and processed around 215,000 particles, had deduced 3D information from these views and also established exactly which part of the molecule was the moving part."
Burgess also remained entrenched in investigating the structure and motion of myosin, but as he points out, dynein research was catching up. Myosin and kinesin were now pretty well understood and thanks to cryo-electron microscopy and direct detector data, researchers could produce high resolution information on the dynein motor.
But as Burgess highlights, researchers in the field were attempting, and often failing, to replicate their experimental methods for myosin and kinesin on dynein.
Instead, and as always, Burgess's approach relied on cryo-electron microscopy with a heavy leaning on image processing. And working with Sutoh and other labs around the world, the researchers finally hit jackpot in 2015.
"Grants came and went, there was a huge amount of image processing and the hardest part was collecting the data," he says. "I was not at all convinced we would ever get this to work, but after six years and against all the odds, my extremely tenacious post-doctoral researcher, Hiroshi Imai, finally got the images."
Freeze etch images of avian spermatozoa: dynein particles line the sides of a microtubule. [Burgess et al]
According to Burgess, their most striking discovery about dynein, was the existence of a hinge between the long thin stalk of the dynein molecule and its grappling hook.
This allows significant variation in the angle of attachment of the motor protein to its track, which, put simply, makes it a great mover.
Thanks to his breakthroughs, work on dynein movement continues. Right now, physicist Dr Sarah Harris along with mathematicians at the University of Leeds have developed a new dynamic 3D model of how macromolecules with large flexible domains move.
"I'd been out of academia twice in my career but came back because I wanted to know how this damn thing worked," laughs Burgess. "I'm now satisfied I've done this and have also captured it in action."
"Persistence has been important but curiosity is key. I think if you're really curious, everything else just falls into place," he adds.
Learn more about Dr Stan Burgess's research here.