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DURHAM, N.C. – Duke University Medical Center scientists have found that the tiny motor molecules within cells "stagger" like humans as they carry their loads of vesicles or organelles through the cell.

By understanding how these intracellular motors work, researchers hope to better understand such diseases as Charcot-Marie-Tooth disease and other neuromuscular conditions dependent on the transport of neurotransmitters or synaptic vesicle precursors by molecular motors.

The researchers, led by cell biologist Sharyn Endow, Ph.D., believe they have helped settle a controversy within biology of how motor molecules, known as kinesins, step through the cytoplasm within the cell along infinitesimal tracks called microtubules.

The motor protein itself consists of a coiled-coil "neck/stalk" region that connects two identical motor domains or "heads." These heads move the molecule forward by binding to the microtubule. Because of these two identical heads, some researchers felt that movement should therefore be symmetrical, one head stepping after the other in an identical fashion.

Endow's research has shown, however, that the molecule alternates which side the lagging head passes the leading head as the motor walks, leading to asymmetrical gait. This alternation is likely due to part of the motor that is itself twisted to the left, the coiled coil stalk.

"Kinesins are fundamental to the proper functioning of the cell, not only as a transporter of vesicles and chromosomes within the cell, but also as an important transmitter of neurotransmitters required to transmit nerve impulses from one cell to another cell," said Endow, who published the results of the Duke research July 15, 2004, in the online edition of the European Molecular Biology Organization (EMBO) journal.

"To date, the exact mechanism by which these molecules travel along microtubules has been poorly understood," she said. "In our experiments, a mutation of a single amino acid produced an exaggerated gait that we believe demonstrates that the kinesin under study walks in an asymmetrical fashion."

Furthermore, Endow continued, the experiments appear to demonstrate the phase at which the breakdown of ATP (adenosine triphosphate) – the source of energy in all cells – powers each "step" of the motor molecule. With each step, the motor breaks down, or hydrolyzes, one molecule of ATP, producing energy and adenosine diphosphate (ADP), but the phase at which the power-stroke occurs had not been previously identified.

It is estimated that there are about 45 different kinesin motors in human. Endow studied a well-characterized kinesin found in fruit flies that closely resembles similar molecules in mammals, including humans.

For their experiments, the researchers induced a mutation in a single amino acid of the kinesin protein. The mutation caused a slight structural change that allowed the motor to more readily release ADP. Researchers then attached microscopic beads to the motors and followed how they moved in a laser trap assay. With this technique, researchers can manipulate single motor molecules by focusing a laser beam through the objective of a microscope.

Under conditions of low force or "load," the mutant motor acted like a normal, or wild type motor – one 8-nanometer step, followed by a pause, and then another 8-nm step, and another pause, as the lagging head moved forward. However, when the mutated motor molecule was exposed to greater force by the laser, the motor took a rapid 8-nm step, followed by a long rest period and slow step, followed by another rapid 8-nm step, followed again by a long rest period and slow step. The rapid steps were faster than the wild type motor, while the slow steps were also slower than those of the wild type.

"This alternating of a slow step with a fast step could be due to the inherent asymmetry of stepping to the right or left of the forward head caused by the handedness of the twist of the coiled coil," Endow said. "Stepping to the right might be constrained by the twist of the coiled coil, while stepping to the left might not be, and might be accelerated by interactions of the rear head with the forward head."

One theory of kinesin movement held that the trailing head always moves in front of the leading head from the same side, lending symmetry to the gait. However, in Endow's model, the trailing head does not always pass the leading head from the same side; instead the two heads alternate and pass to one side or the other of the forward head, leading to an asymmetric gait.

"Since evidence of an asymmetric hand-over-hand model could not be obtained in previous experiments, some scientists felt that an 'inchworm' model made more in sense, in which one head binds to the microtubule and drags the second head behind," Endow continued. "While the inchworm model was consistent with the failure to observe 180-degree rotations that would be expected in the symmetric hand-over-hand models, it did not address the fact that each head hydrolyses ATP to produce force. Therefore, both heads must be involved.

"The model we now favor is asymmetrical," she continued. "The mutant we created enhances the asymmetry and exaggerates what the wild-type motor does."

In addition to diseases like Downs syndrome, in which chromosomes do not divide properly during cell division, deficiencies in kinesins, such as the one studied here, are also implicated in certain neurodegenerative diseases. Finding methods to stimulate kinesin activity might help in the treatment of these diseases. And conversely, methods to inhibit kinesin activity might be a potential approach to slowing down the uncontrollable cell division seen in cancer.

"Our hope is that by better understanding how these motor molecules work we can also understand why sometimes things go wrong in neuromuscular diseases," Endow said. "By using flies as a model, in which the motors are thought to function in a manner similar to that of higher animals, we can understand the motor components and learn how they work in vitro and also in live cells."

The research was funded by the National Institutes of Health, the Japan Ministry of Education, Science, Sport & Culture, the Human Frontier Science Program, the St. Jude Children's Research Hospital Cancer Center, and the American Lebanese Syrian Associated Charities.

Other members of the research team were Hideo Higuchi, Tohoku University, Sendai, Japan; Hee-Won Park, St. Jude Children's Research Hospital, Memphis; and C. Eric Bronner, Duke.