June 11, 2003 CHAMPAIGN, Ill. -- Scientists have developed an extremely accurate imaging technique for looking inside the machinery of a cell and have found that molecules of myosin "walk" in a fashion very much like a human.
"Myosin walks like we walk, but with a 74-nanometer stride that is more than 10 million times smaller than ours," said Paul Selvin, a professor of physics at the University of Illinois at Urbana-Champaign and corresponding author of a paper to appear in the journal Science, as part of the Science Express Web site, on June 5.
Myosin is a tiny molecular motor that converts chemical energy into mechanical motion. While there are more than a dozen types of myosin (including myosin II – the main protein responsible for muscle contractions), Selvin and his collaborators studied myosin V.
"This protein is also responsible for movement," Selvin said, "But not muscular movement. Myosin V is a little cargo transporter in our cells that moves things around by stepping along filaments of actin."
Myosin V is particularly prevalent in nerves. For this reason, mutations in the protein can lead to seizures and other neurological problems. "Like many other biomolecular motors, Myosin V is amazing," Selvin said. "It's tiny, but strong. It can carry more than 1,000 times its own weight."
Myosin V has two "legs" connected to a "body," but exactly how the protein molecule moves its load along helical fibers of actin has been a mystery. "Studies have suggested two main models for movement," Selvin said. "One is the hand-over-hand (or foot-over-foot) 'walking' model in which the two feet alternate in the lead. The other model is the 'inchworm' model in which one foot always leads."
The two models predict significantly different step sizes, Selvin said, but previous imaging techniques have lacked the resolution necessary to measure the step size and determine which model is correct. To measure such minuscule motion, Selvin and his Illinois colleagues -- physics professor Taekjip Ha and graduate students Ahmet Yildiz and Sean McKinney -- developed a single-molecule imaging technique that is capable of locating the position of a fluorescent dye to within 1.5 nanometers. (One nanometer is a billionth of a meter, or about 10,000 times smaller than the width of a human hair). This localization represents a 20-fold improvement over other techniques that use fluorescent dyes.
The researchers also found a way to extend the lifetime of the dye from a few seconds to several minutes. Then they teamed up with physiology professor Yale Goldman and postdoctoral researcher Joseph Forkey, both at the University of Pennsylvania, in applying the technique to measuring myosin movement.
"First, we attach a little fluorescent dye to one of the feet and we take a picture with a digital camera attached to a microscope to find exactly where the dye is," Selvin said. "Then we feed the myosin a little food called adenosine triphosphate, and it takes a step. We take another picture, locate the dye, and accurately measure how far the dye moved."
By looking at the step size, the scientists can tell whether the protein is walking or inchworming along, Selvin said. If it is walking, the rear foot takes twice as big a step than if it were inchworming.
"The long dye lifetime allowed us to measure many consecutive steps, which occurred about once every 3 seconds," Selvin said. "The foot wearing the dye would move forward 74 nanometers, then pause while the unlabeled foot moved forward. The cycle would then repeat itself. The 74-nanometer step size we measured is consistent with a hand-over-hand walking mechanism and inconsistent with an inchworm mechanism."
As an additional check, the researchers also labeled the myosin higher up on the leg, "somewhere in the neighborhood of the thighbone," Selvin said. "The steps alternated in size between long and short, just as you'd expect if it was walking. An inchworm motion, on the other hand, would always yield the same step size, and so we could rule that out."
There are hundreds of different types of biomolecular motors, involved in everything from muscle contraction to moving chromosomes during cell division, to reloading necessary ammunition within nerve cells so they can repeatedly fire. "The cell is a busy place, much like a city where things are constantly moving around," Selvin said. "It will be interesting to see whether all the motors move in the same way."
The work was funded by the National Institutes of Health, the National Science Foundation, the U.S. Department of Energy and the Carver Trust foundation.
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