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Seeing is Believing
stories by Elizabeth Zubritsky
A high-tech lab tunes in to the wilder side of
cells.
In textbooks, there is order. Cell division is precise and scheduled,
with chromosomes marching in formation.
Ted Salmon, professor of biology, knows better. He starts his VCR,
and the television screen fills with writhing, fine-spun tendrils - a
silhouette of Medusa's hair.
"Isn't it great?" exclaims Salmon, watching the tendrils
reach out and retreat. "That's part of the fun I have: being able
to see these 'biological nano-machines' in living cells."
We've been looking at the inner workings of a newt cell magnified
about ten thousand times. Each strand of Medusa's hair is actually a
protein chain, called a "microtubule," that is growing and
shrinking.
Visualizing such things is how Salmon made his mark in cell biology.
He is an engineer-turned-biologist who links image-processing
electronics with microscopes, peering deep
into dividing cells. His video microscopy systems reveal the bustling,
sometimes irregular activities within a live cell - qualities that
static snapshots only imply.
"It's easy to answer the question, 'Do the cells get to this
stage? Do the cells divide?' Ordinary slides can show you that,"
Salmon says. "But when and how do they get there? How orderly is
the process? Those are the questions video microscopy can answer."
And those are the questions that have shaped biologists'
understanding of mitosis, the cell's way of reproducing, and of why the
process sometimes goes awry - causing diseases like Down syndrome or cancer.
Over the past 20 years, Salmon's studies of mitosis have focused on
microtubules, the specialized "rails" that chromosomes move
along, and on kinetochores, patches on the chromosomes where
microtubules attach. He and his students have been able to visualize the
dynamic interactions of structures like these since 1985, when Salmon
built his first system, the VE-DIC (video-enhanced digital interference
contrast) microscope.
The VE-DIC system is sensitive enough to display a single microtubule
in nerve and tissue cells. Actually, the microscope can't focus on a
microtubule, which has a diameter four thousand times smaller than that
of a human hair. Instead, specialized electronics detect the tiny amount
of light scattered by each microtubule and produce a picture. This
technology was developed in the early 1980s by two groups of researchers
working independently. One group was headed by Salmon's former graduate
advisor, Shinya Inoué, then a biology professor at the University
of Pennsylvania.
Salmon and his students used VE-DIC microscopy to study how
microtubules connect to chromosomes and how chromosomes move along
microtubules. They helped confirm a prediction that probability plays a
role in chromosome movement, influencing the growth of microtubules
toward chromosomes (see "Casting for Chromosomes," above) and
the motion of chromosomes once they are connected. As these processes
became clearer, Salmon wanted to learn more about these microtubule
"rails." How strong are they? How flexible?
These questions led him to another tool, the laser optical trap,
which precisely manipulates cellular components. Like a tiny pair of
tweezers, the optical trap's light beam grasps and carries a single
microtubule to another object, such as a kinetochore, held stationary in
the trap. This deliberate movement overcomes the thermal motion,
generated simply by warmth, that randomly scatters objects in a cell.
Salmon has begun this work, using a laser optical trap in Mike Sheetz's
lab at Duke University, and has been awarded $80,000 from the National
Institutes of Health to build his own.
As Salmon has investigated the structural properties of microtubules,
he has also studied their growth. Microtubule fibers grow and shorten at
both ends, even when chromosomes are attached, and this helps move the
chromosomes.
To determine how much
motion each process contributes,
Salmon needed to mark the microtubules and to observe the marks and the
whole array of microtubules simultaneously. He needed a multi-mode
digital imaging microscope, a system he designed and built two years ago.
The multimode digital microscope generates both traditional DIC
pictures and images of fluorescent molecules, which serve as colored
beacons for locating specific proteins or for tracking marks on
microtubules. A CCD (charge-coupled device) - a digital
camera originally designed for astronomy instead of the microcosm of
the cell - captures the images and measures the amount of light
collected. The images can be superimposed, showing the relative
movements of the structures. Changes in light intensity indicate when
proteins have been broken down, produced, or scattered.
Despite the new microscope's utility for some projects, Salmon hasn't
abandoned his old stand-by, VE-DIC. Recently, it allowed two researchers
in the Department of Biology, Kerry Bloom and Elaine Yeh, to discover a
new "checkpoint" during yeast mitosis. Certain checkpoints -
pauses during mitosis when the cell makes sure crucial steps are
complete - have been known for years. But the one found by Bloom and Yeh
occurs at a different stage of mitosis, much later than the others.
VE-DIC microscopy is also non-invasive. More recent techniques such
as fluorescent tagging add foreign materials, some of them toxic, to the
cell.
"You truly can be an observer with VE-DIC," Salmon says,
"and watch the wonderful activity inside a live cell without changing a
thing."
Salmon's work has been supported by the National Institutes of
Health for 20 years. He is a member of the Department of Biology in the
College of Arts and Sciences.
When Cell Division Goes Awry
Down syndrome is one of the most common diseases caused by errors in
mitosis or in meiosis, the closely related process of cell division in
sperm and egg cells.
The disease occurs in one out of every 600 infants and is caused by
the presence of three, instead of two, copies of chromosome 21. Every
month, one to three newborns with Down syndrome are referred to UNC
Hospitals, says Cynthia Powell, assistant professor of pediatrics.
In 95 percent of Down syndrome patients the three copies of
chromosome 21 are separate, as chromosomes normally are. This condition,
called "trisomy 21," arises spontaneously rather than being
inherited. However, having one child with trisomy
21 increases the chance of having another with the condition to 1-2
percent. The risk also increases as a woman ages.
Down syndrome also results from translocations, meaning that the
third copy of chromosome 21 is attached to another chromosome. This
condition, which accounts for 3 percent of the cases, may occur
spontaneously or may be inherited from an unaffected parent. If it is
inherited, the chance that subsequent children also will have a
translocation ranges from 5 percent to 100 percent, depending on the
type of translocation.
Having three sex chromosomes is also possible. These trisomies
usually are less severe than Down syndrome, though they may cause
learning disabilities and problems with socialization. However, the
presence of three copies of most other chromosomes is lethal.
Although the cause of trisomies is unknown, some researchers suspect
that most problems occur in eggs during meiosis. Because eggs are
produced from infancy and may remain in a female for as many as 40
years, some eggs may have a prolonged risk of exposure to factors that
can disrupt cell division.
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