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Modeling myosin minimizes molecular motor mysteries
STANFORD -- When Willi Kuhne discovered the protein myosin in 1864, the German scientist never guessed that it would take his future colleagues more than 120 years to figure out how it drives the movement of all living things.
Now, researchers are getting close, helped by a study that allows them to "see" the crucial protein at work.
In a recent report in Nature, James A. Spudich, Stanford professor of biochemistry and developmental biology, and his collaborators have created a computer model showing the three- dimensional contours of myosin attached to actin, a second protein with which myosin interacts to move cells and contract muscles.
The scientists calculated their model from an electron- microscope image that shows hundreds of these two proteins bound together in an elaborate structure.
"Though the resolution of our model is too small to recognize individual atoms, it provides the three-dimensional shape into which we can fit higher-resolution structures of individual actin and myosin molecules. That allows us to study the interplay of these two proteins in atomic detail," Spudich said.
Fitting these structures into the contour model works well, Spudich said, because myosin is so asymmetric that it slips into the bulges and grooves of the contour model in only one orientation, like a piece of a jigsaw puzzle.
The study was done in collaboration with Rasmus R. Schroder, Werner Jahn and Kenneth C. Holmes at the Max Planck Institute for Medical Research in Heidelberg, Dietmar J. Manstein at Mill Hill University in London, and Hazel Holden and Ivan Rayment at the University of Wisconsin.
While scientists have known since the 1940s that the actin- myosin protein team somehow makes muscles shorten, this study has "implications that go far beyond muscle contraction," a commentator wrote in the July 9 issue of Nature.
In the seventies, scientists spotted actin and myosin in all types of cells, even plants and yeast, where it is involved in the universal process of cell division. During cell mitosis, the protein team constricts the furrow between two dividing cells, gently forcing them apart after all the chromosomes have been sorted out properly.
Actin and myosin also are involved in the heavy traffic that's going on in cells at any moment.
Myosin belongs to a class of proteins known as molecular motors that transport different shipments within the cell. Like a railway system, filaments of actin are laid out in an intricate meshwork of tracks. Along these tracks, the myosin molecules deliver cargo to distant places in the cell. For example, myosin can bind to mitochondria, the tiny cellular power plants, and shuttle them to sites where energy is needed.
At the same time, so-called vesicles, minute storage sacs containing chemicals or cellular waste materials, hitch rides on myosin motors along the actin tracks.
Besides moving particles within cells, molecular motors also can drive whole cells that travel long distances, such as lymphocytes hurrying to the site of an infection or embryonic cells migrating to their final destination in a developing organism.
"The definition of life is movement; molecular motors are that fundamental," Spudich said.
Depicting in atomic detail how actin and myosin carry out these vital functions ideally requires having the crystal structure (a high- resolution image made by shooting X-rays through protein crystals) of actin and myosin bound together in action.
But such crystals are exceedingly difficult to grow, so Spudich and his co-workers turned to the electron microscope and obtained an image of muscle actin and myosin molecules joined together in a regular pattern. With the actin consisting of two thin strands wound around each other and the bulbous myosin molecules sticking out on all sides, that structure looks somewhat like a pipe-cleaner.
Its diameter is only about 200 angstroms (20 billionths of a meter), but that's large enough for some electron microscopy techniques to pick up details.
To improve that microscopic picture, the researchers used a special energy-filtered electron microscope, scanned the resulting image into a computer and refined it with mathematical filtering methods. Then, they computed a model showing its three-dimensional contours - usually a laborious process that involves many slightly different views of an object.
In this case, though, the researchers made use of what Spudich called the "barber pole effect": since it's helical, you only need to look at it from one side to know how it looks from all other sides. The actin- myosin structure is helical and regular, too, and therefore one view comprises all the information about its three-dimensional shape.
"We told the computer to rotate the two-dimensional image 360 degrees and got our three-dimensional model," Spudich said.
Into that helical model, the scientists then fitted the higher- resolution crystal structures of individual actin and myosin molecules to analyze in atomic detail which chemical building blocks of the two proteins, called amino acids, make contact and how they do it.
Supporting this structural analysis is other evidence that Spudich and his coworkers have gathered in previous and ongoing experiments with a primitive slime mold called Dictyostelium discoideum.
Rather than studying complex mammalian cells, Spudich decided to work with a simpler, yet vigorously moving experimental species. His choice fell on this mold, a long-standing favorite of developmental biologists that can undergo a complete transformation of its body.
In forest habitats, Dictyostelium occurs as single cells strewn leisurely across decaying logs, until a chemical signal suddenly rallies these individuals to start wandering about. The cells sniff each other out, and tens of thousands of them stream together to form an amorphous mound. That organism, squishy and about 2 millimeters high, migrates for a while before it bulges upward to form an erect fruiting body.
In the late '80s, Spudich's group proved that myosin performs a vital function for the proper movement and division of these cells by genetically knocking out its gene in Dictyostelium. These disabled mold cells somehow manage to find each other, but linger as a paralyzed lump of about 100,000 cells. That aggregate never migrates or forms a fruiting body.
When his collaborators put a healthy copy of the myosin gene back into the deprived mold, they found that they could rescue all the movements of the previously limp cell lump, thus proving that myosin is essential for that cell mass to change its shape.
Knowing which functions require myosin, Spudich's group started mutating it to learn more about it. By genetically trimming away certain amino acids or replacing some with others, they produce myosin mutants and then check how they affect the mold's motion.
Such experiments teach the researchers which spots in the complex protein are essential for interacting with actin and what areas merely make up the protein's bulk. That information feeds back into the structural analysis reported in Nature.
"We already have about 90 mutations in which we have changed the molecular motor and test how well it still functions," Spudich said. "Some mutations allow perfect movement in Dictyostelium, others completely do the myosin in. A third, interesting class rescues some cell movement but the cells are abnormal. Some are extremely slow, others look like monsters."
Analyzing these mutated myosin proteins biochemically helps the researchers solve the secret of myosin's action: exactly how does this protein operate with actin to make muscles contract and cargo skim through cells?
A widely accepted - but as yet not fully verified - hypothesis holds that the bulbous myosin head attaches at a particular site along the long actin filament, performs an as-yet-mysterious "power stroke" that pulls the actin filament in one direction, lets go of the actin and attaches at another site a little farther downstream to crank the next stroke.
Fueled by adenosine triphosphate (ATP), the cell's energy source, the myosin proteins work their way along the actin filament like a ratchet does, pushing bundles of actin filaments toward each other and in this way contracting the muscle or dividing the cell.
This cycle, though taught in all biology textbooks, is far from proven.
"Nobody understands the actual mechanism by which that power stroke occurs, but there is a very good chance that things will soon fall into place," Spudich said.
"The mutants help us find out. A mutant myosin protein freezes this cycle of events at a particular step, and by knowing the amino acid change of that particular mutant, we can deduce what sites of the protein are responsible for what part of the cycle."
Complicating that picture is a finding that cells contain many different molecular motors, the so-called family of myosin proteins.
"I suspect that each cell has at least 20 molecular motors of the myosin class," Spudich said. Other molecular motor proteins ship neurotransmitters for release at nerve terminals and yet other ATP- driven motors uncoil DNA for replication.
While the scientists have reconstructed the three-dimensional shape of only one kind of myosin, "understanding any one of them in atomic detail will help understand the others, because all molecular motors are likely to be very similar," Spudich said.
All of the different myosin types resemble each other in their bulbous heads, the motor proper, but differ in their tail regions that determine the particular function of each myosin.
These heads are highly conserved throughout the animal kingdom, that is, their amino acid sequences vary very little from one species to another - indicating that the motor system is so essential to life that nature could not afford to change it during evolution.
"Even the myosin of Dictyostelium is 50 percent identical to human myosin. And if you look at the critical spots of the protein, say, where it binds ATP, these are much more conserved than the rest of the protein," Spudich said.
The future prospects of the myosin work look bright, Spudich predicts.
"With the recent convergence of in vitro motility tests, the ongoing mutation studies, the structural biology and new methods of measuring single power strokes of individual protein molecules, the pieces of the myosin puzzle may soon be put together. For us, that is a dream come true."
This story was written by Gabrielle Strobel, a science writing intern at the Stanford News Service
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