Researchers have developed a method
for recording vesicle fusion - The process of neuronal vesicles opening and mixing cargo has been
observed for the first time -
August 18, 2015
Neurons interact with each other via the
release of neurotransmitter molecules but until recently, the actual process
had never been observed. Researchers at
Center for Self-Assembly and Complexity (CSC) and Institute for Basic Science
(IBS) in South Korea have devised a method to observe the release and mixing of
materials between neuronal cell vesicles.
The specific type of cellular structure
they focused on, called vesicles, acts as a vehicle for transporting and
organizing materials within the cell.
Each vesicle is like a tiny, waterproof container that protects the
contents inside of it and keeps its contents separate from the rest of the
cell.

Above:
Illustration of SNARE vesicle fusion process using host-guest FRET pair
When the vesicles need to move material
among themselves, to another organelle or out of the cell, they do it via a
process called vesicle fusion. During the fusion process, the
membranes of the vesicles and the other fusing membrane attach and a small,
secure opening forms that allows material to move between them, (imagine the
way a space shuttle makes a seal when it docks to a space station).
To make this seal, tendril-like fingers
called SNAREs (Soluble N-ethylmaleimide
factor Attachment protein REceptors) on each vesicle find and
pull on each other as they get close to form a tight, intertwining bond. Once this bond is formed, the fusion opening
forms between vesicles and the cargo
they carry can mix.
Until recently, only the result of this
fusion mixing—never the process itself—had been observed. According to first author Bokyoung Gong, “Our
assay with high sensitivity allows an efficient detection of SNARE- mediated
vesicle fusion pore dynamics which have not been observed in previous in-vitro assays.” During experiments, the team discovered that
as many as three open-close cycles (called flickering) happened during the
fusion process. The opening dilated and
constricted during vesicle fusion before the cargo was fully mixed.
To observe this, the team used two different
molecules—one shaped like a hollow pumpkin called cucurbit[7]uril (CB[7]) and
the other a small ball which fits inside of it called adamantane (Ad)—known as
a host-guest pair, that have a high affinity for each other and combining
together. The team made these molecules
each with one half of complementary set of fluorescent dyes which give off one
color when separate and another when combined.
Each dye-conjugated molecule was isolated
in separate vesicles which were then allowed to go through the fusion process
where the contents of each vesicle could combine. Using this method the team was able to see
how the vesicles behaved by observing the color emitted in each of the
individual vesicles before fusion took place and then again when the molecules
combined. No actual neurotransmitter was
mixed during this process, as CB[7]-Ad host-guest pairs were used in its
place. They serve as an easily
observable model for how neurotransmitter and other cargo is mixed between
vesicles.
The observation of the vesicle fusion
mechanism was an important achievement for the CSC and IBS team. Perhaps even more important though is the
method which they devised to make the observations. The CB[7]-Ad host-guest pairs can be used
again in the future for the observation of more than just vesicle fusion.
According to IBS Center for Self-Assembly
and Complexity Director Kimoon Kim, “Such applications should enhance our
ability to study short-lived events in biologically complex processes.” Because of the development of this new
process, scientists may be able able to make observations that were previously
impossible until now. Kim added, “The
power of these probes is demonstrated by the first successful observation of
flickering dynamics of the fusion pore by in vitro assay using neuronal
SNARE-reconstituted vesicles.”
- By
Daniel Kopperud
Notes for editors
-
References
Bokyoung Gong, Bong-Kyu Choi, Jae-Yeol Kim,
Dinesh Shetty, Young Ho Ko,
Narayanan Selvapalam, Nam Ki Lee, and Kimoon
Kim (2015), High Affinity Host−Guest FRET Pair for Single-Vesicle
Content-Mixing Assay: Observation of Flickering Fusion Events, Journal of the
American Chemical Society, DOI: 10.1021/jacs.5b05385
- Media Contact
For further information or to request media
assistance, please contact: Mr. Shi Bo Shim, Head of Department of
Communications, Institute for Basic Science (+82-42-878-8189; sibo@ibs.re.kr)
or Ms. Sunny Kim, Department of Communications, Institute for Basic Science
(+82-42-878-8135;Sunnykim@ibs.re.kr)
-
About the Institute for Basic Science (IBS)
IBS was founded in 2011 by the government of
the Republic of Korea with the sole purpose of driving forward the development
of basic science in Korea It comprises a total of 50 research centers in all
fields of basic science, including mathematics, physics, chemistry, life
science, earth science and interdisciplinary science. IBS has launched 25
research centers as of August 2015.There are eight physics, one mathematics,
six chemistry, eight life science, and two interdisciplinary research centers.
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