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X-rays help researchers reconstruct valuable cellular gateway

Your body is made up of nearly 100 trillion cells that keep you healthy and alive. Each cell has billions of parts of its own, all working in tandem to keep life’s processes moving.

A vital component of a cell is called a nuclear pore, which acts like the doors and windows of a house – it allows important things, like RNA and proteins, to move in and out of the nucleus of a cell. Without nuclear pores, your cells and everything else in your body would shut down. Until now, scientists have not seen exactly how nuclear pores are constructed and how their many parts work.

Enter a team of researchers from the California Institute of Technology (Caltech), led by André Hoelz, professor of chemistry and biochemistry and researcher at the Howard Hughes Medical Institute (HHMI). After almost two decades of perseverance, the researchers succeeded in mapping the atomic structure of the nuclear pore complex (NPC) by determining the structures of its many components and assembling them. Seeing how the NPC interlocks with cells deepens our understanding of how cells work and will potentially lead to new treatments for certain cancers, autoimmune and neurodegenerative diseases, and certain heart diseases.

Unraveling the NPC took time because it’s not a simple puzzle like those waiting in pieces in a box. It contains more than 1,000 individual proteins, and it can take scientists years to map just one before they even begin to assemble them. The whole process is like a gigantic three-dimensional puzzle, but made up of pieces so tiny you can’t see them with the naked eye or even the best optical microscope.

To make this step possible, the Caltech team turned to high-energy X-rays generated by the Stanford Synchrotron Radiation Light Source (SSRL) at the Department of Energy’s SLAC National Accelerator Laboratory (DOE). ), the advanced photon source at the DOE’s Argonne National Laboratory. , and National Synchrotron Light Source II at the DOE’s Brookhaven National Laboratory. In numerous experiments over the years, they have zapped samples of crystallized NPC proteins with X-ray light, illuminating the atomic structure and general shape of the samples. They published their findings this month in two articles in Science. The first paper reported on the architecture of the face that sits outside the core, and the second paper revealed how the many parts of the NPC are held together by “glue” proteins.

“X-ray crystallography provided atomic detail on individual protein components,” said SLAC senior scientist Aina Cohen. “As technologies improved, including at SLAC’s SSRL, researchers were able to see the complex of nuclear pores more clearly, so they could put the different proteins together to complete this complex puzzle.”

Without SSRL’s improved technology over the years, such as its microfocusing capabilities and a pixel array detector (PAD), installed in 2009, the research could not have taken place, Hoelz said. SSRL had one of the first PADs in the country, and the detector generated much better X-ray diffraction data than before, helping Caltech researchers map the protein structures of the NPC. Determining the crystal structure of a chunk of six proteins and identifying its arrangement in the nuclear pore in 2015 showed that with patience and diligence, researchers could eventually provide a complete picture of the entire NPC. .

“SSRL was the facility where most of the initial structural work took place due to the wide access we had via the Caltech Molecular Observatory, an X-ray crystallography facility with access to the 12-2 beamline of SSRL,” Hoelz said. “This regular access allowed for the systematic improvement of various aspects of X-ray diffraction experiments, allowing us to solve even the most difficult nucleoporin structure determination problems. We had several structures on which we have worked for over a decade before solving them.”

The completed human NPC puzzle will provide a framework upon which many important experiments can now be performed, said Christopher Bley, senior postdoctoral researcher in chemistry at Caltech and also co-first author of the studies.

“We now have this composite structure, and it enables and informs future experiments on NPC function, or even disease,” Bley said. “There are a lot of mutations in the NPC that are associated with terrible diseases, and knowing where they are in the structure and how they come together can help design the next set of experiments to try to answer questions about what What are these mutations doing?

Having determined the structure of human NPCs, scientists can now focus on working out the molecular basis of various enigmatic functions of NPCs, such as how mRNA is exported, the underlying causes of the many associated diseases to NPCs and the targeting of NPC function by many viruses, including SARS-CoV-2 and the monkeypox virus, in an effort to develop new therapies, Hoelz said.

SSRL, APS, and NSLS-II are DOE Office of Science user facilities. The research was funded by HHMI, the National Institutes of Health and the Heritage Medical Research Institute.

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