Researchers have developed a method for recording vesicle fusion

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.