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Needling Chromosomes Yield Insights into Cell Division

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Duke Health News 919-660-1306

DURHAM, N.C. -- 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 that is 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.

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 the A. S. Pearse
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 parting of 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," Niklas 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 aggravated by us and indebted to us," he said.

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