Understanding how proteins tighten that garrote-like filament around the membrane “neck” of the cell may be of great value because the basic mechanism is shared by retroviruses, including HIV, that take over cells to reproduce. Once the squeeze is on, other proteins that use the filament like a scaffold jump into action, cutting the membrane neck and re-fusing the two membranes.
The so-called ESCRT-III complex is just one of the many machines that make life as we know it possible. When linked together, the Vps32 oligomer forms a single-stranded, expanding spiral that attaches itself to the membrane within the cell.
The ESCRT-III membrane scission machine is made of Vps32 proteins (or CHMP4B in humans) and assembles into spiral, spring-shaped filaments. Individual spirals were highlighted in a variety of colors by researchers in this cryo-electron micrograph.
“These spirals are generated with a very unique architecture where the filaments within the spiral are carefully spaced, which allows it to assemble in a spring-like conformation,” said Jon Audhya, associate professor of biomolecular chemistry at the UW School of Medicine and Public Health. “Like a spring, when you stretch it out, it has some potential energy to it. When you change the organization of that spring, it can collapse back down and expel that energy, and we think something very similar is happening with Vps32 spiral filaments.”
The filament undergoes a dramatic change when the Vps4 protein connects to it. Once connected, the spiral structure contracts, bringing the cell membrane with it and closing the gap at this final stage of division. The filament collapses down to about 20-25 nanometers in diameter.
But the filament not only draws the membrane together; it’s also scaffolding other proteins onto the site. Audhya hypothesizes that those proteins act downstream to ultimately drive the separation and fusion of the membrane.
To discover how this filament works, Audhya and collaborators from across campus used nearly every strategy available to understand the process, including cryo-electron microscopy, structural modeling approaches, theoretical computational modeling, and in vivo models with yeast and worms.
“We spanned the entire spectrum of those approaches and I think that comprehensive strategy is really the only way we’re going to understand how these proteins assemble,” said Audhya. “It was really fortuitous that we had all this expertise here on campus, and it was a tremendous collaboration between five laboratories, lots of post-docs, grad students and technical work that came together.”
The striking images of the filaments were produced through cryo-electron microscopy, rapidly freezing the purified proteins in liquid ethane so fast that ice crystals can’t form, also known as vitrification. Once they had enough of the Vps32 (CHMP4B is the human analog), the protein was laid on a grid and vitrified. The cryo-electron microscopy and other work was published in a recent issue of the Journal of Cell Biology, and an image from the study was featured on the cover.
“When there is organization present like these spirals, they become pretty apparent even with the limited contrast offered by cryo-EM, and we can use those structures and computationally try to understand how the individual monomers fit together to form this protein machine,” he said.
The understanding of how this machine assembles will be fundamental in understanding how it works in all instances of a scission event, whether it’s in retrovirus replication and secretion, vesicle release or cytokinesis.
In a translational context, understanding the assembly process of Vps32 may someday allow scientists to modulate the system, perhaps attacking problems like HIV budding. HIV hijacks the host cell’s membrane to package, protect, and spread viral progeny.
“We’re not going to come up with the drug necessarily, but now that we have this model, other people can attack that question in a way that they couldn’t before this paper,” said Audhya.
This is believed to be the first publication of a biological sample from UW’s recently acquired cryo-electron microscope that’s been done completely on campus. Other lab groups that were part of this discovery include the labs of Marisa Otegui, professor of botany and genetics; Paul Ahlquist, professor of oncology and virology and investigator of the Howard Hughes Medical Institute; Qiang Cui, professor of chemistry, and Julie Mitchell, associate professor of biochemistry and mathematics. Qingtao Shen, a postdoctoral fellow working in the Audhya lab, was responsible for most of the electron microscopy work in this study.
University of Wisconsin School of Medicine and Public Health