Decoding the Nuclear Pore Complex of the Cell

Whatever you're doing, whether driving a car, going for a jog, or even sitting on the sofa munching chips and watching TV, a full suite of molecular machinery is hard at work within each of your cells. That machinery, which is far too tiny to be seen with the naked eye or with many microscopes, generates energy for the cell, builds proteins, copies DNA, and does a lot more.

The nuclear pore complex is one of those pieces of equipment, and it's one of the most complicated (NPC). The NPC is a highly discriminating gatekeeper for the cell's nucleus, which is a membrane-bound area inside a cell that houses the cell's genetic material. It is made up of over 1,000 unique proteins. On its route in or out of the nucleus, everything must pass via the NPC.

The NPC's position as a nucleus gatekeeper makes it crucial to the cell's operations. DNA, the cell's permanent genetic information, is transcribed into RNA within the nucleus. The RNA is subsequently taken out of the nucleus to be employed in the production of the proteins that the cell requires. The NPC ensures that the nucleus has the ingredients it requires to synthesize RNA, as well as shielding the DNA from the harsh environment outside the nucleus and allowing the RNA to escape the nucleus after it has been produced.

“It’s a little like an airplane hangar where you can repair 747s, and the door opens to let the 747 come in, but there’s a person standing there who can keep a single marble from getting out while the doors are open,” says AndrĂ© Hoelz, a Caltech professor of chemistry and biochemistry and a Howard Hughes Medical Institute Faculty Scholar. Hoelz has been investigating and interpreting the structure of the NPC in connection to its function for more than two decades. He has methodically chipped away at its secrets over the years, piece by piece, piece by piece, revealing them.

The ramifications of this study might be enormous. The NPC is not only crucial to the cell's activities, but it also plays a role in a variety of disorders. Some incurable malignancies, neurological and autoimmune disorders including amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalopathy, and heart problems like atrial fibrillation and early abrupt cardiac death are caused by mutations in the NPC. Moreover, many viruses, like the one that caused COVID-19, target and shut down the NPC as part of their lifecycle.

Hoelz and his colleagues highlight two major discoveries in a pair of publications published in the journal Science: the determination of the structure of the NPC's outer face and the explication of the process by which specific proteins work as a molecular glue to keep the NPC together.

A very tiny 3D jigsaw puzzle

Hoelz and his colleagues describe how they mapped the structure of the side of the nuclear pore that faces outward from the nucleus and into the cells' cytoplasm in their publication "Architecture of the cytoplasmic face of the nuclear pore." To accomplish so, scientists used imaging methods such as electron microscopy and X-ray crystallography on each puzzle piece to solve the equivalent of a very small 3-D jigsaw puzzle.

The process began with Escherichia coli bacteria (a type of bacteria commonly used in labs) that were genetically engineered to produce the proteins that make up the human NPC, according to Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the papers' co-first authors.

“If you walk into the lab, you can see this giant wall of flasks in which cultures are growing,” Petrovic explains. “We express each individual protein in E. coli cells, break those cells open, and chemically purify each protein component.”

Once the purification was complete—which may take up to 1,500 liters of bacterial culture to produce enough material for a single experiment—the research team began methodically testing how the NPC's components fit together.

Rather of dumping all of the proteins into a test tube at once, the researchers evaluated pairings of proteins to determine which ones would fit together like two jigsaw pieces, according to George Mobbs, a senior postdoctoral scholar research associate in chemistry and another co-first author of the work.If a pair of proteins fit together, the researchers would test them against a third protein until they found one that did, and then the resulting three-piece structure would be tested against additional proteins, and so on. Working their way through the proteins in this manner finally yielded the final outcome of their paper: a 16-protein wedge that is repeated eight times to form the NPC's face, similar to pizza slices.

“We reported the first complete structure of the entire cytoplasmic face of the human NPC, along with rigorous validation, instead of reporting a series of incremental advances of fragments or portions based on partial, incomplete, or low-resolution observation,” says Si Nie, postdoctoral scholar research associate in chemistry and co-first author of the paper. “We decided to patiently wait until we had acquired all necessary data, reporting a humungous amount of new information.” 

Martin Beck of the Max Planck Institute of Biophysics in Frankfurt, Germany, and his colleagues utilized cryo-electron tomography to create a map that revealed the contours of a jigsaw into which the researchers had to arrange the parts. Hoelz and Beck shared data over two years ago to speed up the conclusion of the riddle of the human NPC structure, and subsequently constructed structures of the full NPC separately. “The substantially improved Beck map showed much more clearly where each piece of the NPC—for which we determined the atomic structures—had to be placed, akin to a wooden frame that defines the edge of a puzzle,” Hoelz adds.

The Hoelz group's empirically established architectures of NPC components contributed to confirm the Beck group's models. “We placed the structures into the map independently, using different approaches, but the final results completely agreed. It was very satisfying to see that,” Petrovic says.

“We built a framework on which a lot of experiments can now be done,” says Christopher Bley, a senior postdoctoral scholar research associate in chemistry and co-first author on the paper.  “We have this composite structure now, and it enables and informs future experiments on NPC function, or even diseases. 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.” 

“This elegant arrangement of spaghetti noodles”

The other paper, titled "Architecture of the linker-scaffold in the nuclear pore," describes how the research team determined the entire structure of the NPC's linker-scaffold—the collection of proteins that help hold the NPC together while also allowing it to open and close and adjust itself to fit the molecules that pass through.

Hoelz compares the NPC to a set of Lego bricks that don't lock together but are held connected by elastic bands that keep them basically in place while enabling them to wander about.

“I call these unstructured glue pieces the ‘dark matter of the pore,'” Hoelz explains. “This elegant arrangement of spaghetti noodles holds everything together.” 

The technique for determining the structure of the linker-scaffold was similar to that used to determine the structure of the other elements of the NPC. The scientists produced and purified huge amounts of the various types of linker and scaffold proteins, examined individual connections using a number of biochemical studies and imaging techniques, then evaluated them piece by piece to see how they fit together in the whole NPC.

They tested their findings by making mutations in the genes that code for each of the linker proteins in a live cell. They could anticipate what would happen to the structure of the cell's NPCs when those faulty proteins were injected since they understood how those mutations would modify the chemical properties and form of a certain linker protein, rendering it dysfunctional. They knew they had the proper arrangement of linker proteins if the cell's NPCs were functionally and structurally deficient in the way they predicted.

“A cell is much more complicated than the simple system we create in a test tube, so it is necessary to verify that results obtained from in vitro experiments hold up in vivo,” Petrovic explains.

The completion of the NPC's outer face also contributed to the resolution of a long-standing conundrum surrounding the nuclear envelope, the double membrane system that surrounds the nucleus. The nuclear membrane, like the cell membrane inside which the nucleus dwells, is not entirely smooth. Integral membrane proteins (IMPs) are molecules that serve a number of functions, including functioning as receptors and aiding in the catalysis of biological events.

Although IMPs may be found on both the inner and outer borders of the nuclear envelope, how they go from one side to the other has remained a mystery. Because IMPs are locked inside the membrane, they are unable to pass via the NPC's primary transport channel like free-floating molecules.

Once Hoelz's team established the structure of the NPC's linker-scaffold, they recognized that it allowed for the production of small "gutters" around its outer edge, which allow IMPs to pass through the NPC from one side of the nuclear envelope to the other while remaining entrenched in the membrane.

“It explains a lot of things that have been enigmatic in the field. I am very happy to see that the central transport channel indeed has the ability to dilate and form lateral gates for these IMPs, as we had originally proposed more than a decade ago,” Hoelz adds.

The findings of the two articles, taken combined, constitute a significant advance in scientists' knowledge of how the human NPC is created and functions. The findings of the researchers pave the way for considerably further investigation.  “Having determined its structure, we can now focus on working out the molecular bases for the NPC’s functions, such as how mRNA gets exported and the underlying causes for the many NPC-associated diseases with the goal of developing novel therapies,” Hoelz says.