|Decoding a key cell
Whatever you're doing, whether you're driving a car, walking, or even at your laziest, eating potato chips and watching TV on the couch, there's a whole host of molecular machines inside every single one of your cells working hard.
This machine, too small to be seen with the naked eye or even with several microscopes, generates energy for the cell, makes its proteins, makes copies of its DNA, and much more.
Among those pieces of machinery, and among the most complex, is something known as a nuclear pore complex (NPC).
The NPC, made up of more than 1,000 individual proteins, is an incredibly distinctive gatekeeper to the cell nucleus, the membrane-bound region within a cell that contains that cell's genetic material. Anything entering or exiting the kernel must pass through the NPC on its way.
The role of the NPC as a gatekeeper to the nucleus means that it is vital to cell processes. Within the nucleus, DNA, the permanent genetic code of a cell, is transcribed into RNA. This RNA is then taken out of the nucleus so that it can be used to make proteins that the cell needs.
The NPC ensures that the nucleus gets the materials it needs for RNA synthesis, while also protecting the DNA from the harsh environment outside the nucleus and enabling the RNA to leave the nucleus after its synthesis.
"It's a bit like a hangar where you can fix 747s, and the door opens to let the 747 in, but someone standing there can block a single marble from coming out while the doors open," says André of Caltech. Hoelz, professor of chemistry and biochemistry and faculty scientist at Howard Hughes Medical Institute.
Hoelz has been studying and deciphering the structure of the NPC in relation to its function for over two decades. Over the years, he has steadily torn her secrets, revealing them piece by piece.
The implications of this research are likely to be huge. Not only is NPC central to cell processes, but it is also involved in many diseases.
Mutations in NPC are responsible for some intractable cancers, neurodegenerative and autoimmune diseases such as amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalitis, and for heart diseases including atrial fibrillation and sudden early cardiac death.
Additionally, many viruses, including the virus responsible for COVID-19, target and shut down the NPC during their life cycle.
Now, in a pair of papers published in Science, Hoelz and his team describe two important discoveries: defining the structure of the outer face of an NPC and elucidating the mechanism by which special proteins such as the molecular glue act to hold the NPC together.
Very small 3D puzzle
In their paper, "Cytoplasmic Face Geometry of Nuclear Pores," Holz and his research team describe how they mapped the structure of the side of the NPC that faces outward from the nucleus into the cytoplasm of cells.
To do this, they had to solve the equivalent of a very small 3D puzzle, using imaging techniques such as electron microscopy and X-ray crystallography on each puzzle piece.
Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the research's co-authors, says the process began with Escherichia coli (a strain of bacteria commonly used in laboratories) that was genetically modified to produce the proteins that make up the human NPC.
"If you go into the lab, you can see this giant wall of the flasks in which the cultures grow," Petrovich says. "We express each individual protein in the Escherichia coli cells, break down those cells, and chemically purify each protein component."
Once this purification was complete — which can require up to 1,500 liters of bacterial culture to get enough material for a single experiment — the research team began rigorous testing of how the NPC pieces fit together.
George Mobbs, a postdoctoral researcher involved in chemistry and another co-author of the paper, says that the assembly occurred in a "gradual" fashion; Instead of pouring all the proteins together into the researchers' test tube at the same time, the researchers simultaneously tested the pairs of proteins to see which ones could fit together, like two pieces of a puzzle.
If a pair that fits together is found, the researchers would then test the two now fused proteins against a third protein until they find one that fits in that pair, then test the resulting three-piece structure against the other proteins, and so on.
Working their way through the proteins in this way eventually led to the end result of their paper: a 16-protein wedge that, like slices of pizza, repeats eight times to form an NPC's face
“We have reported the first fully cytoplasmic facet structure of a human NPC, along with rigorous validation, rather than a series of progression,” says Si Ni, a postdoctoral researcher in chemistry who is also a co-first author of the paper.
Intrinsic fragments or fragments based on partial, incomplete, or low-resolution observation. “We decided to wait patiently until we gained all the necessary data, and to report a massive amount of new information.”
Their work complemented research by Martin Beck of the Max Planck Institute for Biophysics in Frankfurt, Germany, whose team used cryo-electron tomography to create a map that provides the contours of the puzzle in which the researchers had to place the pieces.
To speed up the completion of the human NPC skeleton puzzle, Hoelz and Beck exchanged data over two years ago and then built the entire NPC skeletons independently. "The greatly improved Beck map showed more clearly where each piece of the NPC - for which we identified atomic structures - would be located - similar to a framework," says Hoelz. Wooden defines the edge of the puzzle.
The experimentally determined structures of the NPC plots from the Hoelz group served to validate the modeling by the Beck group. "We mapped the structures independently, using different approaches, but the end results totally agreed."It was gratifying to witness," Petrovich says.
"We've built a framework on which a lot of experiments can now be done," says Christopher Bley, a postdoctoral research associate in chemistry and co-first author. “We have this synthesized structure now, and it enables and informs future experiments about NPC function, or even diseases.
There are a lot of mutations in NPCs that are associated with terrible diseases and figuring out where they are in the structure and how they occur. Together they can help design the next set of experiments to try to answer questions regarding what these mutations do.”
"This elegant arrangement of spaghetti noodles"
In another paper, titled "Nuclear Pore Junction Scaffold Engineering," the research team describes how they outlined the entire structure of what's known as the NPC linker scaffold — the group of proteins that help hold NPCs together while also providing them with the flexibility they need to open, close and adjust themselves to fit molecules that pass through. across it.
Hoelz likens the NPC to something built of Lego bricks that fit together without locking together, instead of being held together by rubber bands that mostly keep them in place while allowing them to move around a bit.
"I call these unstructured bits of gum 'pore dark matter,'" says Holz. "This elegant arrangement of spaghetti noodles ties everything together."
The process for characterizing the linker scaffold structure was largely similar to that used to characterize other parts of the NPC. The team synthesized and purified large quantities of many types of linker and scaffold proteins, used a variety of biochemical experiments and imaging techniques to examine individual interactions, and tested them piece by piece to see how they fit together in intact NPC.
To verify their work, they inserted mutations into the genes that code for each of those binding proteins in the living cell. Because they knew how these mutations would change the chemical properties and shape of a particular binding protein, making it defective, they could predict what would happen to the cell's non-playable structure when those defective proteins were introduced. If the cell's NPCs were functionally and structurally defective in the way they expected, they knew they had the correct arrangement of the linker proteins.
“The cell is much more complex than the simple system we make in a test tube, so it is necessary to verify that the results obtained from in vitro experiments hold up in vivo,” Petrovic says.
The assembly of the outer face of the NPC also helped solve an ancient mystery about the nuclear envelope, the double-membrane system that surrounds the nucleus. Like the cell membrane in which the nucleus is located, the nuclear membrane is not completely smooth. Instead, it is studded with molecules called integrated membrane proteins (IMPs) that function in a variety of roles, including acting as receptors and helping to catalyze biochemical reactions.
Although IMPs can be found on both the inner and outer sides of the nuclear envelope, it has not been clear how they actually travel from one side to the other. Indeed, because IMPs are stuck within the membrane, they cannot only glide through the central transport channel of the NPC as free-floating molecules do.
Once Hoelz's team understood the structure of the NPC linker scaffold, they realized that it allowed the formation of tiny "gutters" around its outer edge that allow IMPs to slide through the NPC from one side of the nuclear envelope to the other while always remaining embedded in the same membrane.
“This explains a lot of Things that were mysterious in this area. I am very pleased to see that the central transmission channel really has the potential to expand and form side gates for these engines, as we originally proposed over a decade ago,” says Holz.
Taken together, the results of the two papers represent a leap forward in scientists' understanding of how the human NPC is constructed and how it functions. The team's findings open the door to further research. "Having determined its structure, we can now focus on establishing the molecular bases for NPC functions, such as how mRNA is exported and the underlying causes of many NPC-related diseases with the goal of developing new therapies," says Holz.
Papers describing the work appear in the June 10 issue of Science.
The research was funded by the National Institutes of Health, the Howard Hughes Medical Institute, and the Heritage Institute for Medical Research.
Christopher J. Bley et al. Architecture of the cytoplasmic face of the nuclear pore. Science, 2022 DOI: 10.1126/science.abm912
Stefan Petrovic, Dipanjan Samanta, Thibaud Perriches, Christopher J. Bley, Karsten Thierbach, Bonnie Brown, Si Nie, George W. Mobbs, Taylor A. Stevens, Xiaoyu Liu, Giovani Pinton Tomaleri, Lucas Schaus, André Hoelz. The architecture of the linker scaffold in the nuclear pore. Science, 2022; 376 (6598) DOI: 10.1126/science.abm9798