Cells say the darndest
things: Chemists uncover individual messages
BY LILA GUTERMAN
In an important early step
toward understanding the chemistry of human thought,
Stanford chemists have managed for the first time to read
the individual chemical messages that cells exchange.
These messages account for
nearly all of the communication that takes place in the
brain, but previous efforts to record them one at a time
have been thwarted by their extremely small size. More
than a billion vesicles the microscopic membrane-bound
pouches that carry the chemical signals could fit into
an average-sized drop of water.
"We really are seeing
a new era dawning in which people are trying to
understand the chemistry of the brain and of the central
nervous system, with the possibilities of amazing
consequences, from treating mental disease to improving
mental powers," says Richard N. Zare, who headed the
research effort.
Zare, the Marguerite Blake
Wilbur Professor of Chemistry, and his colleagues
published the technique that allowed them to read out the
contents of individual vesicles in the journal Science
earlier this year.
Cells use vesicles to
signal each other in a variety of ways. Such messages
include mail to the brain that says "ouch!"
from a bumped funny bone. Other messages regulate the
female reproductive cycle.
By analyzing large numbers
of vesicles together, scientists had learned a great deal
about the vocabulary of the cell's language. But this
method can deduce only average messages. It is as if
researchers knew that in 1,000 letters, the word
"food" appeared 1,800 times, but they could not
discern how many times (if at all) "food"
showed up in any one letter.
Zare suspected the average
message might not be a good representation of any single
vesicle's contents. "Could it be that the real
signals are being hidden and confused by looking at only
averages?" he wondered. "I thought likely so,
and indeed our work shows that to be the case."
The signal from a single
vesicle is important because cells send and receive
vesicles one at a time. "The contents of one vesicle
are enough to stimulate a cell," says Richard
Scheller, professor of molecular and cellular physiology
at Stanford and a Howard Hughes Medical Institute
investigator. He and Zare have been working toward
analyzing single cellular packages for several years.
"Almost all
[communication] within our brains is the result of
vesicles being released one at a time," Scheller
says. "Different responses are elicited from
vesicles with different contents. So it's very important
to know the contents of the individual vesicles."
But the vesicles that
transmit messages between brain neurons are too tiny to
manipulate and examine. Instead, Zare and Scheller chose
to work with the vesicles that regulate egg-laying in sea
slugs. These vesicles are about 1,000 times larger than
those the brain uses large enough to analyze with
Zare's new technique. "Snails don't have many cells,
but the cells they have are large and juicy," Zare
explains.
Daniel Chiu, a graduate
student, and Sheri Lillard, a postdoc in Zare's lab,
developed the technique, which captures one vesicle at a
time, pops it open and analyzes its contents.
The first step in this
process is to trap a single vesicle and introduce it into
a tiny chamber, where Chiu and Lillard can manipulate it.
This step is not as simple as it may sound. Because the
vesicle is so small, the researchers have to use delicate
procedures.
The chemists trap the tiny
package using a laser. The vesicle moves to the most
intense area of the laser beam and stops there. Once the
vesicle is immobilized, the researchers move a minuscule
tube next to the vesicle. A slight electric current
slides the vesicle into the tube.
Next, the researchers add
chemicals that tear open the package and label its
contents with a dye. This dye, which attaches only to
specific molecules, glows when light hits it. The dye
transforms the vesicle's chemicals into beacons.
The dye fastens to several
different molecules perhaps all the nouns in the
cellular message but the researchers want to know how
much of each compound was in the vesicle. So Chiu and
Lillard separate the mixture into its components by
forcing the chemicals to move along the tiny tube that
contains them.
Because each different
chemical moves at a characteristic speed, the various
compounds arrive at a detecting station at different
times. The detecting station watches for the lights to
pass, a brighter glow indicating a larger amount of one
chemical, a dimmer light meaning a smaller quantity. In
this way, Lillard and Chiu measure the relative amounts
of the various chemicals.
The chemists then identify
each chemical compound in collaboration with researchers
led by two former postdoctoral researchers from Zare's
lab: Owe Orwar, a chemist at Chalmers University in
Göteborg, Sweden, and Evan R. Williams, a chemistry
professor at the University of California-Berkeley. The
researchers can thereby read the cellular mail.
Using this technique, the
researchers have found that individual vesicles have
varying contents, even though the vesicles came from the
same gland. "One vesicle would have one component
but it would be completely absent from another
vesicle," Lillard says. "We knew that in a
population, both components were present, but when we got
down to the level of a single vesicle, there were clear
differences."
But what does this
variability mean for the sea slug? "I can summarize
that in three words," says Zare. "I don't know.
Not yet. Is it the difference between mature and immature
vesicles? Is it the difference of some control? I don't
know."
It's one thing to read
individual words in cellular mail; it's another problem
altogether to translate the language. But simply reading
the chemical codes that the vesicles contain is an
important step toward understanding cellular
communication, the researchers maintain.
Eventually, Zare and
Scheller hope to extend the technique to look at the
contents of smaller vesicles, such as those released by
neurons in the brain.
Zare says, "I think
we are showing the way, developing it step by step, so
that we can apply this to smaller systems and, indeed,
learn about the chemical basis of thought."
The research was funded in
part by the National Institute on Drug Abuse and the
National Science Foundation. SR
Lila Guterman is a
Stanford News Service intern.
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