Stepping Back in Time

Figuring out how molecular motors can parade, step by step, through living cells has required research by many determined scientists over several decades. In September, the Lasker Foundation honored the contributions of three of those researchers with the 2012 Albert Lasker Basic Medical Research Award: HHMI investigator Ronald Vale of the University of California, San Francisco, Michael Sheetz of Columbia University, and James Spudich of Stanford University School of Medicine.

The shared prize reflects the importance of research that occurred in the space of a few years during the 1980s, when the three researchers’ paths intersected at Stanford University and the Marine Biological Lab in Woods Hole, Massachusetts. The findings that each made during this time changed the way scientists could study molecular motors.

“There’s very little doubt that this short time between 1982 and 1986 was key,” says Spudich. “We finally did all the right experiments.”

The story of motor proteins begins before that, however. In 1954, Sir Andrew Huxley of the University of Cambridge published a paper hypothesizing that the contraction of muscles relies on two types of muscle filaments sandwiched together that slide in opposite directions. At the same time, researcher Hugh Huxley (no relation), then at the Massachusetts Institute of Technology, used electron microscopy to get the first pictures of muscle fibers. His images supported the “sliding filament” theory—the filaments of actin and myosin could be seen moving past one another. Hugh Huxley took his observations a step further, proposing that myosin was the molecule that caused movement, exerting force on the actin filaments (see Observations, “Really Into Muscles”).

Spudich joined the research effort as a postdoctoral fellow in Hugh Huxley’s lab and continued to focus on muscle when he established his own lab at Stanford in 1977. His goal: to figure out a way to prove Huxley’s theory and study myosin motors one molecule at a time. His lab probed the moving parts inside a plethora of cell types, even some from organisms without obvious muscles, like molds and algae. When his team turned to the slime mold Dictyostelium, they hit pay dirt, finding a way to isolate both actin and myosin from the cells.

“We were tremendously excited about the possibilities these results presented as a small step along the way to an in vitro motility assay,” writes Spudich in a recent Nature Medicine paper reflecting on the history. While the molecules isolated from Dictyostelium didn’t immediately provide proof of the sliding filament model of muscle contraction, they did help reveal the importance of motor proteins, showing, for example, that cell division relies on them.

By the time Sheetz joined Spudich’s lab in 1982—for a sabbatical from his own work—the scientists were eager to develop a way to study the movement outside of cells. They suspected that previous attempts to watch myosin move on actin filaments had been thwarted by the fact that the filaments weren’t lined up in the same direction. But they knew of an organism—the freshwater alga Nitella—that had naturally aligned actin filaments.

“By the time the ’80s came, it wasn’t that there was a shift in culture or in our overall approach to science,” says Spudich, “It was just that we had generated more and more knowledge about actin and myosin that allowed us to try new things.”

So with their knowledge about Nitella and the idea that directionality mattered in the movement of myosin, Spudich and Sheetz began splitting open cells of the alga. Since they wanted to preserve alignment of the filaments, they performed experiments directly on the splayed cells, adding myosin bound to plastic beads that helped them follow its movement.

“The ‘Eureka!’ moment was that it worked the very first time we tried it,” says Spudich. “We immediately knew this approach would work if we could clean it up.”

As the team fine-tuned the assay, it became clear that the movement of myosin—which, they discovered, relied on the cellular energy molecule ATP—matched up with rates of movement seen in filaments during muscle contraction.

By this time, Vale was a graduate student at Stanford and looking for a research project to focus on.

“Spudich and Sheetz were right downstairs from me,” says Vale, “and just being around that research was fascinating. So I decided to see if the movement they were seeing had a possible connection to the movement inside nerve cells.”

Sheetz—who was returning to Woods Hole at the end of his sabbatical with Spudich—became interested in Vale’s question, and they came together at the Marine Biological Lab to work on the topic. They began looking inside the huge nerve cells of the squid, using a form of light microscopy (video-enhanced differential interference contrast microscopy), and saw a similar movement of molecules taking place. But when they looked closer and isolated the motor proteins, they discovered that the moving molecules weren’t actin and myosin. Instead, they were a different motor protein, one that walked along microtubules. By 1985, Vale and Sheetz had isolated the new motor protein, dubbed kinesin, and set up an assay similar to Spudich’s actin-myosin system, showing that kinesin walks in one direction along microtubules.

“That was really a second ‘Eureka!” moment,” says Spudich. “Finding out that there’s a whole new motor system.”

In that span of years during the early 1980s, article after article was published adding to the story of how molecular motors work. Spudich’s lab went on to show that the motor domain of myosin alone could move along actin, and researchers Bob Allen, Scott Brady, and Ray Lasek developed videos showing the movement of entire organelles inside squid cells.

In the coming decades, Vale, Sheetz, and Spudich continued delving into the dynamics of the motor proteins. But it was their early efforts at developing these assays, which are still used today, that drove the study of molecular motors as well as the field of single-molecule research.

“Even in those early days, it seemed clear that motors must be doing an awful lot of things in the cell,” says Sheetz. “But I don’t think any of us would have imagined the full extent of it. We just wanted to figure out a way to study these systems.”

-- Sarah C.P. Williams
HHMI Bulletin, Winter 2013

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