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Needling chromosomes yields insights into cell division

23.12.2004


By impaling individual chromosomes with glass needles one thousandth the diameter of a human hair, a Duke University graduate student has tested their "stickiness" to one another during cell division. Her uncanny surgical skills have added a piece to the large and intricate puzzle of how one cell divides into two -- a process fundamental to all organisms.

In the Dec. 14, 2004, issue of Current Biology, Leocadia Paliulis and Bruce Nicklas report their progress in understanding how the pairs of chromosomes in each cell manage to balance their adhesion to one another and their release during cell division. Their work was sponsored by the National Institutes of Health. Chromosomes are the tiny fiber structures in the cell that house its genes. They replicate and separate in the process of cell division.

The exquisite management of adhesion properties between newly divided chromosomes, called chromatids, is crucial if the cells are to divide properly. In this process chromatids are drawn apart to separate poles of the dividing cell so that each new "daughter" cell contains a single copy of each. The same basic process operates in normal cell division, called mitosis, as well as the proliferation of sperm and egg cells called meiosis.



"Chromosomes in mitosis and meiosis have to be held together, because otherwise they don’t attach to the apparatus called the spindle that distributes them to opposite poles," explained Nicklas, who is a Research Professor of Biology. "If they’re held together, then one replicated chromatid can attach to one pole and the other to the opposite pole. But if they are not held together, they attach independently, and often both sister chromatids can go to the same pole rather than to opposite poles. This creates chromosome imbalances that can lead to cancer or chromosomal abnormalities that cause birth defects."

According to Nicklas, it was known that the two sister chromatids adhered to one another and released at the appropriate time during cell division. However, that understanding was based on biochemical experiments that revealed when the "glue" protein called cohesin that holds chromatids was degraded during cell division. Also, microscopic studies had shown that there appeared to be two separate chromatids during an early stage of cell division, so it was believed that they had detached from one another at that time. "What hadn’t been done was to attempt to separate chromatids to directly determine whether they, in fact, are held together or not," said Nicklas. "So, Leocadia set out to use micromanipulation to distinguish between visible separateness and physical separateness."

To study chromatid adhesion, Paliulis mastered the high art of manipulating two infinitesimal glass needles to impale each of two sister chromatids in cultured grasshopper cells at the appropriate time in cell division. Then, she would ever-so-gently apply force to pull them apart. Upon release, if they remained apart it revealed they were separated; but if they snapped back together the researchers would know the chromatids were still attached. Paliulis was a Duke graduate student when she performed the experiments, but is now a postdoctoral fellow at the University of North Carolina at Chapel Hill

Paliulis’s skill in the task was extraordinary, said Nicklas. "First of all the needles are invisible in the cell, so you have to continually move them back and forth to detect their position by how they disturb structures around them. Also, the micromanipulation apparatus is arranged such that your view is up through the bottom of the cell, and the needles are coming down through a layer of oil covering the cell to preserve it.

"So, you also have to constantly adjust the focus to determine where the needle is coming down into the cell. This is difficult with one needle, but with two it’s a terrific challenge; and you really need an almost tactile sense of where the needles are." Nicklas said that even the smallest misstep could result in broken needles, stretched chromatids or ripped-apart cells. However, he said, Paliulis mastered the delicate technique and performed numerous experiments pulling the chromatids apart at different points along their length and at different times during cell division.

The experiments revealed that the chromatids are attached to one another, but that they initially separate at their centers, zipping apart until they are entirely separate. Then, they can be drawn to the opposite poles of the dividing cell. The experiments also revealed that it is the erosion of linkages between the chromatids, and not any tension exerted by the spindle, that causes the chromatids to separate.

Also intriguing, found the researchers, was that the chromatids mysteriously remained stuck to one another at a time when biochemical analysis could not detect any cohesin proteins in the cell. Nicklas believes that the twin chromatids may still have some entanglements between the corresponding DNA strands on each chromatid. DNA, which makes up genes in the cell, replicates itself as a central process in chromosome duplication.

"So, we’re left with the mystery of what molecules hold the chromatids together at this point in cell division," Nicklas said. "But that’s the usual outcome of work in my laboratory and a sign that we’re doing good science since we raise new questions. We lay the mechanistic groundwork for the molecular explanations that have to be made. So, our colleagues who do molecular work are both provoked and challenged by us," he said.

Dennis Meredith | EurekAlert!
Further information:
http://www.duke.edu

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