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Biology, the aerial view
To understand the processes of life we must get past the school buses, says Sandy Simon
BY JOSEPH BONNER
Proteins in our bodies’ cells have
always been believed to zip around the cytoplasm, wriggling in and
out of the nucleus and escaping through the cell membrane as they
perform the array of tasks that keep us alive and well. Recently
scientists have visualized these dances in the cell by fusing a
cellular protein to a fluorescent tag from jellyfish. This has led
to many insights into the cell, but comes with many cautionary
tales.
“Our eyes catch the brightest star in
the heavens — the signals from a cell that shine
strongest,” says Sandy Simon, professor and head of the
Laboratory of Cellular Biophysics at Rockefeller. It is a classic
example of what microscopists call the “yellow school
bus” problem.
“Let’s say you’re a spy and
you come to America to figure out what is the function of the
yellow school bus,” says Simon. “I give you a spy
plane, and you fly over the country taking pictures of all the
school buses. What you mainly get are pictures of large parking lots filled with school buses.
Every so often you’ll see a single school bus on the street,
but obviously it’s a rare, stray event. You conclude
it’s not significant.”
Scientists studying protein transport in
living cells too often have tended to see only the school bus
depots — the locations where proteins are synthesized. When
they see something that associates with the protein they assume
that it is an important co-factor or chaperone when in reality it
may a nearby but unrelated structure.
“In biochemistry and genetics, when you
look at this macroscopic picture — focusing on 10,000 buses
and ignoring two — you’re averaging,” explains
Jyoti Kumar Jaiswal, a postdoctoral fellow in the Simon lab. By
averaging, Jaiswal says, scientists may be missing important events
that are happening inside the cell.
“The way around the conundrum is to
observe the single school buses,” says Jaiswal.
“It’s the one that is actually performing the function it
was built for: picking up and dropping off children. With patience,
eventually you’ll see that every bus leaves the
depot.”
(Of course, single events can also be
misleading. “Each single event you observe is an
anecdote,” Simon says. “Let’s say you were an
alien spying on Earth, and you happened to pick Adolph Hitler as
your subject to follow. The impression you get of the human race
might not be the same as if you chose Winston Churchill.”)
Simon’s lab tackles the yellow school
bus problem with the biophysicist’s version of the spy plane:
a special fluorescence imaging system called TIRFM, or total
internal reflection fluorescence microscopy. Using TIRFM,
scientists can track a protein of interest with a fluorescent tag
and follow it as it travels near the cell membrane. Three important
discoveries have recently emerged from the Simon lab’s
efforts to focus on a single bus.
How vesicles unload their cargo
A family of proteins called synaptotagmins was
widely believed to trigger the release of a cell’s cargo. But
after using TIRFM to study the function of a specific synaptotagmin
protein, SytVII, Simon, Jaiswal and their colleagues say it
actually restricts release.
SytVII is one of 15 members of the
synaptotagmin family of fusion proteins, which are associated with
vesicles that contain hormones and nerve signaling proteins. In
response to a rise in the level of calcium in the cell,
synaptotagmins are believed to help trigger fusion of the vesicles
with the cell membrane allowing them to release their contents, a
process called exocytosis.
Jaiswal expected vesicles in SytVII-deficient
cells to fail to release their cargo, or at least to delay it. But
the exact opposite occurred: vesicle release was faster and more
robust. The absence of SytVII caused the vesicles to release even
cargo that would otherwise have been too big to pass through the
fusion pore.
“The faster response to calcium argued
against the assumption that SytVII was doing its job as a
calcium-dependent trigger for fusion,” says Jaiswal.
The findings, published in the August issue of PLoS Biology,
help to explain how cells heal potentially lethal tears in their
membranes by using cellular structures called lysosomes —
regulated in part by SytVII.
“This finding may explain Jyoti’s
previous discovery in which he found that when a cell has a rupture
in its surface the membrane will try to patch the surface by fusing
lysosomes there, but preventing the intermixing of the lysosomal
and plasma membrane proteins,” says Simon. “Syt VII may
be a scaffold that holds all those membrane proteins together, so
they can’t diffuse out, and the cell has a way of putting a
Band-Aid on to heal over.”
How SNAREs help membranes fuse
The secretion of insulin by a pancreatic cell,
the transmission of a signal by a brain cell, and the growth of all
types of cells depend on the proper fusion of cell membranes. In a
May issue of Proceedings of
the National Academy of Sciences, Simon,
Jaiswal and postdoc Marina Fix set out to answer once and for all
the question: what role do a class of membrane proteins called
SNAREs play in membrane fusion?
According to Simon, cell biologists are divided
into two camps: one that has argued that SNAREs are the key
proteins necessary for membranes to fuse, and the other countering
that SNAREs need to dock with vesicles before fusion can occur.
“To answer this question, we decided to
start from scratch, build up a system and ask ‘What does the
cell need for fusion to occur,’” says Simon.
First, Fix recreated the plasma membrane by
forming a “lipid bilayer” on the coverslip of a
microscope slide. She then added two types of SNAREs, vesicle-based
(v-SNAREs) and target-membrane (t-SNAREs), and found that they did
not fuse to the bilayer. But when she added calcium to the mix, in
a concentration designed to mimic what occurs naturally at the
synaptic terminal of nerve cells in the body, she watched as
individual vesicles released their contents as they fused to the
lipid bilayer.
“Marina’s findings are the first
solid evidence that SNAREs are the minimal sufficient machinery to
get physiologically relevant fusion,” says Simon.
Jaiswal says that the PNAS paper validates
the Simon lab’s approach. “Our experiments used
reagents that have been used by other researchers in many other
experiments; it’s not that we made these things up ourselves.
The difference is that other studies were done as an average, in
populations. No one looked at single vesicles.”
Watching as tumors glow
Proteins aren’t the only biological
structures that can be tracked by using fluorescence. Sandy
Simon’s lab was one of the first to use quantum dots,
nano-sized fluorescent crystals, to simultaneously track multiple
proteins in living cells for days at a time. Now, in research
reported in the September issue of Nature Medicine,
Simon’s lab extended this technique to track cancer tumor
cells in living mice.
Because the glow they emit is bright, long
lasting and highly precise, quantum dots overcome some of the
obstacles that limit the usefulness of other organic fluorescent
molecules. Simply by altering their size, scientists can
manufacture them to produce light in any color of the rainbow, and,
additionally, only one wavelength of light is required to
illuminate all of the different-colored dots. Thus, spectral
overlap doesn’t limit the number of colors that can be used
at once in an experiment. In addition, quantum dots shine for an
average of 1,000 times longer than most known fluorescent dyes.
In the Nature
Medicine paper, Simon, Jaiswal and
postdoc Evelyn Voura injected melanoma tumor cells tagged with
quantum dots into the tail veins of mice. Using a two-photon
microscope at Rockefeller’s Bio-Imaging Resource Center, they
tracked the tumor cells as they metastasized and traveled to the
lungs. The tumor cells labeled with quantum dots were equally
proficient as the unlabeled cells in metastasizing and forming
tumors, and the cells did not have any detectable effect on the
health of the mice.
The Rockefeller researchers say that quantum
dot labeling of tumor cells could have an enormous impact on how
scientists everywhere investigate the mechanisms of metastasis.
“The ability to specifically tag cells with quantum dots can
be a crucial tool that could enable oncologists to monitor
quantitatively how well cancer cells have been eradicated after
experimental treatments,” says Jaiswal.
November 19, 2004
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