Scientists are deciphering the nuclear pore complex with incredible detail. Credit: Valerie Altunian
Many of us have learned the basic cell structure at some point and will remember components such as the cell membrane, cytoplasm, mitochondria and nucleus. However, the structure of our cells is actually much more complex than you might think. In fact, because we have discovered so much over the years, we now know that cells are much more complex than even biologists have realized long ago.
One element of particular complexity is the nuclear pore complex. The eukaryotic cell nucleus surrounds a double membrane, the nuclear envelope that encloses the genetic material of the cell nucleus. This nuclear envelope encompasses the complex of nuclear pores, which, although microscopic in size, is an incredibly complex molecular machine consisting of a huge number of different proteins.
Whatever you do, whether you’re driving a car, jogging or even the laziest, eating chips and watching TV on the couch, there’s a whole range of hard-working molecular machines in each of your cells. This machine, too small to be seen with the naked eye or even with many microscopes, creates energy for the cell, produces its proteins, makes copies of its DNA and much more.
Among these parts of machines and one of the most complex is something known as a nuclear pore complex (NPC). An NPC, which is made up of more than 1,000 individual proteins, is an incredibly discriminatory gatekeeper for the cell’s nucleus, the membrane-bound region inside the cell that holds the genetic material of that cell. Everything that enters or leaves the kernel must pass through the NPC on its way.
Molecular model of the outer (cytoplasmic) surface of the nuclear pore complex. Reprinted with permission from CJ Bley et al., Science 376, eabm9129 (2022). Credit: Hoelz Laboratory / Caltech
The role of the NPC as the gatekeeper of the nucleus means that it is vital for cell operations. Within the nucleus, DNA, the constant genetic code of the cell, is copied into RNA. This RNA is then removed from the nucleus so that it can be used to produce the proteins the cell needs. NPC ensures that the nucleus receives the materials it needs to synthesize RNA, while protecting DNA from the harsh environment outside the nucleus and allowing RNA to leave the nucleus after it has been made.
“It’s a bit like an airplane hangar where you can repair a 747 and the door opens for the 747 to come in, but there’s someone standing there who can stop a marble from coming out while the doors are open,” said Andre of Caltech. Holtz, Professor of Chemistry and Biochemistry and Fellow of the Howard Hughes Medical Institute. For more than two decades, Holz has studied and deciphered the structure of the NPC in relation to its function. Over the years, he has constantly unraveled his secrets, revealing them piece by piece.
The consequences of this study are potentially huge. NPC is not only central to cell operations, but is also involved in many diseases. Mutations in NPCs are responsible for some incurable cancers, neurodegenerative and autoimmune diseases such as amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalopathy, as well as cardiac conditions, including atrial fibrillation and early sudden cardiac death. In addition, many viruses, including those responsible for COVID-19, target and exclude NPCs throughout their life cycle.
Now, in a couple of articles published in the magazine science, Holtz and his research team described two important discoveries: determining the structure of the outer surface of NPCs and elucidating the mechanism by which special proteins act as a molecular glue to hold NPCs together.
Very small 3D puzzle
In their paper entitled “Architecture of the Cytoplasmic Surface of the Nuclear Pore”, Holtz and his research team describe how they mapped the structure of the NPC side, which faces outward from the nucleus and into the cytoplasm of cells. To do this, they had to solve the equivalent of a very small 3-D puzzle using imaging techniques such as electron microscopy and X-ray crystallography on each piece of the puzzle.
Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the co-authors of the reports, says the process began with E. coli bacteria (a strain of bacteria commonly used in laboratories) that have been genetically engineered to produce the proteins that make up the human NPC.
“If you walk into the lab, you can see this giant wall of flasks where crops grow,” says Petrovich. “We express every single protein E. coli cells, break down these cells and chemically purify each protein component. “
Once this purification – which may require up to 1,500 liters of bacterial culture to produce enough material for one experiment – was completed, the research team began diligently testing how the NPC pieces fit together.
George Mobs, a senior postdoctoral fellow in chemistry and another co-author of the paper, says the gathering happened in a “step-by-step” way; instead of pouring all the proteins together in a test tube at the same time, the researchers tested pairs of proteins to see which ones would fit together, like two pieces of a puzzle. If a pair is found that fits together, the researchers will test the two now combined proteins against a third protein until they find one that matches that pair, and then the resulting three-part structure was tested against other proteins, and so on. Passing them through the proteins in this way eventually led to the end result of their paper: a wedge of 16 proteins that was repeated eight times, like slices of pizza, to form the face of the NPC.
“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 increasing advances in fragments or parts based on partial, incomplete, or low-resolution observations,” said Si Nie, a postdoctoral researcher. in chemistry and also co-author of the article. “We decided to wait patiently until we had all the necessary data, announcing a huge amount of new information.”
Their work complements research conducted by Martin Beck of the Max Planck Institute for Biophysics in Frankfurt, Germany, whose team uses cryoelectron tomography to generate a map that outlines the puzzle in which researchers had to place the pieces. To speed up the completion of the puzzle of the structure of the human NPC, Hoelz and Beck exchanged data more than two years ago and then independently built structures for the entire NPC. “Beck’s substantially improved map showed much more clearly where each piece of NPC should be placed – for which we determined the atomic structures – similar to a wooden frame that defines the edge of the puzzle,” says Holtz.
The experimentally determined structures of NPC pieces from the Hoelz group served to validate the modeling from the Beck group. “We put the structures on the map independently, using different approaches, but the end results were completely in agreement. It was very satisfying to see that, “said Petrovich.
“We have built a framework on which many experiments can now be done,” said Christopher Blee, a senior postdoctoral fellow in chemistry and co-author. “We currently have this composite structure and it enables and informs future experiments on the function of NPCs or even diseases. There are many mutations in NPCs that are associated with terrible diseases, and knowing where they are in the structure and how they come together can help develop the next set of experiments to try to answer the questions of what these mutations do. ”
“This elegant arrangement of spaghetti noodles”
In another article, entitled “Linker Scaffold Architecture in the Nuclear Pore,” the research team describes how it determined the entire structure of what is known as the NPC linker scaffold, the collection of proteins that help keep NPCs together while it provides the flexibility it needs to open and close and adjusts to fit the molecules that pass through.
Hoelz likens the NPC to something made of Lego bricks that fit together without locking, and instead are tied together with rubber bands that keep them mostly in place while allowing them to move around a bit.
The nuclear pore complex (NPC) is able to expand and contract to adapt to the needs of the cell. Reprinted with permission from S. Petrovic et al., Science 376, eabm9798 (2022). Credit: Hoelz Laboratory / Caltech
“I call these unstructured pieces of glue the ‘dark matter of the pores,'” says Holtz. “This elegant arrangement of spaghetti noodles holds it all together.”
The process for characterizing the structure of the linker scaffolding was almost the same as the process used to characterize the other parts of the NPC. The team produces and purifies large quantities of many types of linker and skeletal proteins, uses various biochemical experiments and imaging techniques to study individual interactions and tests them piece by piece to see how they fit together in an intact NPC.
To test their work, they introduced mutations into the genes that encode each of these linker proteins in a living cell. Because they knew how these mutations would change the chemical properties and shape of a specific linker protein, making it defective, they could predict what would happen to the cell’s NPC structure when these defective proteins were introduced. If the cell’s NPCs were functionally and structurally defective in the way they expected, they knew they had …
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