Nearly 100 trillion cells make up your body, which keeps you alive and in good health. The processes of life are maintained by the billions of pieces that each cell has on its own.
Nuclear pores, one of the most important parts of a cell, function like the windows and doors of a house by allowing significant substances, such as RNA and proteins, to enter and exit the nucleus of a cell.
Your cells and the rest of your body would stop working if nuclear pores weren’t present. Scientists have not previously been able to determine the precise composition and operation of nuclear pores.
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).
By figuring out the architectures of its numerous parts and fitting them together, researchers finally succeeded in mapping the atomic structure of the nuclear pore complex (NPC) after nearly two decades of perseverance.
Understanding how the NPC functions within cells advances our knowledge of how cells function and may help develop new treatments for several malignancies, autoimmune and neurological disorders, as well as some heart issues.
Because the NPC is not a straightforward puzzle like those that are waiting in pieces in a box, unraveling it required some time. It has more than 1,000 distinct proteins, and it might take years for researchers to map just one of them before they can start putting them together. The entire process resembles a massive three-dimensional jigsaw puzzle, but one built of pieces that are so small that neither the best light microscope nor the human eye can see them.
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.
Professor André Hoelz
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.
They have used X-rays to illuminate the atomic structure and general shape of crystallized NPC protein samples in several experiments throughout the years. This month, they presented their findings in two papers published in Science.
The architecture of the face on the outside of the nucleus was described in the first study, and the second paper described the “glue” proteins that hold the NPC’s numerous components together.
“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.”
The research would not have been possible without SSRL’s enhanced equipment throughout the years, such as its microfocus capabilities and a pixel array detector (PAD), installed in 2009, according to Hoelz.
The detector produced far better X-ray diffraction data than was previously achievable, assisting the Caltech researchers in mapping the protein structures of the NPC. SSRL was home to one of the nation’s earliest PADs.
It was demonstrated in 2015 that, with perseverance and diligence, the researchers could eventually provide a comprehensive picture of the entire NPC by determining the crystal structure of a sizable six-protein chunk and determining its arrangement in the nuclear pore.
“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 HHMI, National Institutes of Health, and the Heritage Medical Research Institute funded the research.
