Contact: Ian Clark, (608) 890-5641, iclark@uwhealth.org
MADISON, Wis. — From fertilizing an egg to a virus entering into a cell, to hormones moving into a synapse, cell membranes are almost constantly fusing together. The first step in membrane fusion is the creation of a pore that forms the first connection between the two membranes destined to fuse, but what that actually looks like was unclear — until now.
Using a variety of approaches, including the use of nanodiscs, University of Wisconsin-Madison researchers have gained valuable insight into the structure and regulation of fusion pores — the initial connection between two separate cell bodies. That connection ranges in size depending on the membranes to be fused, but is usually just one or two nanometers in diameter. For comparison, a sheet of paper is roughly 100,000 nanometers thick.
In 2013, the Nobel Prize was awarded to a group of investigators who identified the proteins that start and manage most membrane-fusion reactions. This crucial work has armed researchers with the molecules that mediate fusion, but it’s unclear how many of these proteins operate. For example, it’s well-known that two types of proteins called SNAREs assemble together into helical bundles that form the core of fusion machinery. Without these proteins, membrane fusion doesn’t work, but how that machinery works remained unclear.
Studying fusion pores is incredibly challenging for several reasons. Not only are these pores very small, they are very short-lived; they are open for only a few thousandths of a second. To examine the structure of this fusion engine in progress, Ed Chapman — a Howard Hughes Medical Institute professor in the neuroscience department at the UW School of Medicine and Public Health — and a Chapman lab postdoc, Huan Bao, turned to nanotechnology.
Nanodiscs are tiny disc-shaped patches of membranes built by researchers to help study membrane proteins. The two types of discs used in the fusion pore study, merely six and 13 nanometers in diameter, were used to collar the pores. The pores would open through the nanodisc, but the discs wouldn’t allow the pore to expand, seemingly freezing this split-second reaction in time.
“The beauty of using nanodiscs to study fusion pores is the fact that they are bounded by membrane scaffolding proteins,” said Chapman. “Once the fusion pores open, they can’t dilate because the nanodisc can’t expand, trapping fusion pores in their initial open state, thus making it possible to directly study them at the biochemical and structural level for the first time.”
This work resolves a long-standing conundrum in the field: do SNAREs act to pull bilayers together to form purely lipidic pores, or are the pores lined by SNARE proteins? These experiments, published in Nature Structural and Molecular Biology, revealed that they are hybrid structures, composed of both lipids and SNARE proteins.
The Chapman lab now seeks to study the kinetic properties of these reconstituted fusion pores, and to use the nanodisc system to gain even more detailed insights into their structure.
