Duke Study Helps Explain How Cells Divvy Up Genes During Reproduction

In a report published in the Aug. 21 issue of the journal Science, Duke cell biologist Sharyn Endow and postdoctoral fellow Kimberly Waligora said they have for the first time been able to take apart a molecular motor, put it back together, make it run in reverse, and then mutate the motor and make it run the other way.

The motor they worked on is a key component of the machinery that divides up the genetic material during a special type of cell division that makes sperm and eggs. When this machinery malfunctions, chromosomes, the carriers of genetic material, can become jumbled and the wrong number of chromosomes can end up in each egg. This can result in infertility or syndromes such as Down's, in which a child has an extra copy of chromosome 21.

The scientists hope these experimental models will give clues to why some women have multiple miscarriages, and why, as women age, more of their eggs have the wrong number of chromosomes, increasing the odds of Down's syndrome and other birth defects.

Endow discovered the motor, called Ncd (nonclaret disjunctional), in fruit flies, but similar motors operate in all animals, including people, she said.

"Our hope is that by understanding how these molecular motors operate, we will be able to identify why sometimes things go wrong in the reproductive process," Endow said. "Right now it is very difficult to do these experiments with animals more advanced than flies because they make eggs internally. That makes it difficult to observe the process. But using flies, in which the process is thought to be closely related to higher animals, we can identify the components and learn how they work."

The research was funded by a grant from the National Institutes of Health.

Over the past decade, scientists have discovered dozens of these tiny molecular motors inside cells. Ncd belongs to a family of motors called kinesins. These highly efficient devices are responsible for shuttling cargo along a highway system made of microscopic tubes called microtubules inside the cell. Kinesin motors do everything from delivering chemical messages to helping cells divide.

But Endow's motor, Ncd, was odd. It seemed to run in reverse. All the other discovered kinesins shuttled cargo in one direction along the microtubules. Ncd went the other way. Yet when scientists looked at the motors in detail, they seemed to be identical.

Endow and Waligora decided to find out how the motor worked by taking it apart and putting it back together. They studied the structure of the motor and identified several distinct parts: the "engine," the "neck," and the "stalk." The engine generates the power for the motor, but the roles of the neck and stalk were a mystery.

Using molecular biology techniques, the scientists created a hybrid motor that has a kinesin engine and a neck and stalk section from Ncd. This hybrid motor runs in reverse, just like Ncd. The scientists therefore concluded that the neck and stalk section must determine the direction the motor runs.

They further tested their theory by making a mutation in the neck, in effect "breaking" the neck. This motor reverted to the forward direction.

"For the first time, we have been able to identify a motor component that is responsible for determining the direction and helping coordinate motor movement," Endow said. "This is a crucial initial step in finding out how molecular motors help organize chromosome segregation during cell division."

To help visualize just how the Ncd motor organizes the apparatus responsible for dividing up chromosomes equally during cell division, Endow and her colleagues attached a glowing fluorescent molecule to the Ncd motor in fruit flies. Then they photographed the chromosomes and molecular motors in action as the chromosomes lined up to separate during cell division, producing some of the first detailed moving pictures of chromosomes being divvied up into individual eggs.

They then made movies of normal cell division and what happens when Ncd is not working properly. When the scientists mutated Ncd, the cell division apparatus doesn't form properly, and chromosomes don't end up divided evenly among the resulting eggs. A reports on this research, detailing how the Ncd motor participates in the cell division process that creates eggs, appears in the September issue of the Journal of Cell Science. The movies can be viewed on the Internet at http:/abacus.mc.duke.edu/moviepage.html.