S T A N F O R D M E D I C I N E

Volume 16 Number 4, SUMMER 1999


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published quarterly by Stanford University Medical Center, aims to keep readers informed about the education, research, clinical care and other goings on at the Medical Center.

 

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splitting images

 

BY KRISTIN WEIDENBACH


AN ANIMAL CELL READY TO REPRODUCE STARTS BY DUPLICATING JUST TWO OF ITS COMPONENTS -- THE DNA THAT CARRIES THE CELL'S GENETIC INFORMATION AND AN UNASSUMING LITTLE STAR-SHAPED CELL BODY CALLED THE CENTROSOME. Scientists have known for many years how DNA duplicates but they are only just beginning to learn how the centrosome makes a copy of itself. * The centrosome has a critical role in cell division. When a cell prepares to divide, the centrosome duplicates and the two freshly minted sibling centrosomes migrate to opposite ends of the cell. A web of fibers connects the two centrosomes, forming a spindle upon which the chromosomes gather at the cell center. When the spindle is complete, the chromosomes split in half and retract along the spindle fibers toward the centrosomes located at the poles of the cell. A nuclear membrane forms around each chromosomal clump and eventually two new cells are born ­ each with an identical set of chromosomes carrying the full complement of genes.

Working together, scientists in the laboratories of Peter Jackson, PhD, assistant professor of pathology and of microbiology and immunology, and Tim Stearns, PhD, assistant professor of biological sciences and of genetics, have now found that the centrosome begins to duplicate when a pair of even smaller bodies inside the centrosome splits into two. The splitting apart of these tiny cylindrical bodies -- the centrioles -- is the first in a series of steps that culminates in an animal cell dividing into two perfect replicas. The ringmaster of this intricate performance is a protein complex called cyclin E/Cdk2.

Cyclin E/Cdk2 was already known to play a crucial role in DNA replication but its influence over centrosome division was not understood until the Stanford team and another group from the University of Massachusetts Medical School announced their findings. The Stanford scientists described their research in the March 16, 1999, issue of the Proceedings of the National Academy of Sciences; the Massachusetts group's research is in the February 5, 1999, issue of Science.

The two teams used a similar experimental approach in their investigations, with a jelly-like mass of frog eggs playing a key role in both. The eggs are large and divide rapidly following fertilization, making them a perfect vehicle for studying the early events of cell division. "We can use frog eggs to serve as a little test tube," says Jackson.

The Stanford team used the eggs to show that cyclin E/Cdk2 causes the centrosome to divide. By fiddling with the eggs they could make a centrosome replicate wildly in the presence of cyclin E, producing up to eight centrosomes per cell, instead of the usual two. When they added an inhibitor of cyclin E, the centrosome sat sedately and refrained from dividing.

To confirm the crucial role of cyclin E/Cdk2 they delved deeper into the centrosome and studied the tiny centrioles housed inside. Most of the time these two bundles of fibers sit serenely side-by-side, but in a cell that is preparing to divide, the bundles move apart -- the first sign that cell division is about to occur.

Using fluorescent microscopy and colored fluorescent dyes the researchers were able to look inside the centrosome and see the centrioles as two tiny dots. When they incubated the centrosomes in cyclin E/Cdk2, the researchers saw that the two dots separated, indicating that the twin centrioles had split apart. When the researchers added the cyclin E inhibitor, the two dots remained tightly coupled.

The results of this test clinched the finding that the first step of centrosome duplication is the separation of the centrioles and that initiation of this event requires cyclin E/Cdk2. But the researchers continued to wonder about what holds the centrioles together until cyclin E/Cdk2 dictates that they should come apart.

"The image would be that you have some sort of glue that holds these things together and you have to dissolve the glue in order to get them to separate," says Jackson.

In the September 1, 1999, issue of Genes and Development, the researchers reveal what they have learned about how this "glue" operates. A protein called Skp1 orchestrates the dissolution of the glue by tagging the "glue protein" for destruction. Once the glue protein is removed by protein-destroying enzymes, the centrioles separate and DNA replication begins.

The presence of so many checkpoints in the centrosome replication cycle highlights its importance to the process of error-free cell division. Having the correct number of centrosomes is critical for accurate cell division, according to the researchers. If there are too many centrosomes, an abnormal three- or four-cornered spindle will form and the chromosomes will be pulled in random directions when the cell begins to divide. Daughter cells can end up with too many or too few chromosomes, resulting in gain or loss of genetic information.

Multiple centrosomes and abnormal numbers of chromosomes are characteristic of cancer cells, and the Stanford team believes that defects in the control of centrosome duplication are an obvious mechanism by which cells with an abnormal number of chromosomes can be produced.

"If you don't set up the spindle properly, then the chromosomes are not going to end up in the right place," says Jackson. "It's clear that some cancers lose chromosomes at a high rate and that chromosome loss may be a reflection of problems in this mechanism." SM