X-rays help researchers piece together treasured cellular gateway

One vital component of a cell is called a nuclear pore, which acts like the doors and windows in a house -- they allow important things, like RNA and proteins, to enter and exit a cell's nucleus. 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 built and how their many parts function.

Enter a team of researchers from the California Institute of Technology (Caltech), led by André Hoelz, professor of chemistry and biochemistry and faculty scholar of the Howard Hughes Medical Institute (HHMI). After almost two decades of persistence, researchers successfully mapped the atomic structure of the nuclear pore complex (NPC) by determining the structures of its many components and fitting them together. Seeing how the NPC fits together in cells furthers our understanding about how cells work and will potentially lead to new treatments for certain cancers, autoimmune and neurodegenerative diseases, and certain heart conditions.

Unraveling the NPC took time because it's not a simple puzzle like the ones that wait in pieces in a box. It contains more than 1,000 individual proteins, and it can take scientists years to map a single one before they can even begin to put them together. The entire process is akin to a gigantic three-dimensional jigsaw puzzle, but one that is made from pieces so tiny that you cannot see them with your naked eyes or even with the best light microscope.

To make this milestone possible, the Caltech team turned to high-energy X-rays generated by the Stanford Synchrotron Radiation Lightsource (SSRL) at the Department of Energy's (DOE) SLAC National Accelerator Laboratory, 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 many experiments over the years, they zapped crystallized NPC protein samples with X-ray light, illuminating the samples' atomic structure and overall shape. They published their findings this month in two papers in Science. The first paper reported the architecture of the face that lies at the outside of the nucleus, and the second paper revealed how the many pieces of the NPC are held together by "glue" proteins.

"X-ray crystallography provided atomic details of the individual protein components," Aina Cohen, SLAC senior scientist, said. "As technologies have been improving, including at SLAC's SSRL, researchers have been able to see the nuclear pore complex in clearer ways, so that they could fit the different proteins together to complete this complex puzzle."

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

"SSRL was the facility where most of the initial structural work occurred due to the ample access we had through Caltech's Molecular Observatory, an X-ray crystallography facility with access to SSRL's Beam Line 12-2," Hoelz said. "This regular access allowed for the systematic improvement of various aspects of the X-ray diffraction experiments, which allowed us to solve even the most challenging nucleoporin structure determination problems. We had multiple structures that we worked on for over a decade before we solved them."

The completed human NPC puzzle will provide a framework on which a lot of important experiments can now be done, said Christopher Bley, a senior postdoctoral scholar research associate in chemistry at Caltech and also co-first author of the studies.

"We have this composite structure now, and it enables and informs future experiments on NPC function, or even diseases," 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 and answer the questions of what these mutations are doing."

Having determined the human NPC structure, scientists can now focus on working out the molecular basis for various enigmatic functions of NPCs, such as how mRNA gets exported, the underlying causes for the many NPC-associated diseases, and the targeting of NPC function by many viruses, including SARS-CoV-2 and monkeypox virus, with the goal of developing novel therapies, Hoelz said.

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