Scientists are deciphering the nuclear pore complex in incredible detail. Credit: Valerie Altounian
Many of us 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 complicated than you might think. In fact, since we’ve been discovering a lot of things over the years, we now know that cells are much more complex than even biologists even realized not long ago.
An element of particular complexity is the nuclear pore complex. Around the nucleus of the eukaryotic cell is a double membrane, the nuclear envelope, which closes the genetic material of the cell nucleus. The complex of nuclear pores that encompasses this nuclear envelope, which although microscopic in size, is an incredibly complex molecular machinery made up of a large number of different proteins.
Whatever you do, whether it’s driving a car, going for a run, or even in the laziest time, eating chips and watching TV on the couch, there’s a whole bunch of molecular machinery inside each of your cells. · Cells in effort. That machine, too small to see with the naked eye or even with many microscopes, creates energy for the cell, makes its proteins, makes copies of its DNA, and more.
Among these pieces of machinery, and one of the most complex, is something known as the Nuclear Pore Complex (NPC). NPC, which is made up of more than 1,000 individual proteins, is an incredibly discriminating gateway to the nucleus of the cell, the region attached to the membrane within a cell that contains the genetic material of that cell. · Lula. Anything that enters or leaves the core must pass through the NPC in its path.
A molecular model of the outer (cytoplasmic) face of the nuclear pore complex. Reprinted with permission from CJ Bley et al., Science 376, eabm9129 (2022). Credit: Hoelz / Caltech Laboratory
The role of the NPC as a nucleus gatekeeper means that it is vital to cell operations. Inside the nucleus, DNA, the permanent genetic code of the cell, is copied into RNA. This RNA is then carried out of the nucleus so that it can be used to make the proteins that the cell needs. The NPC ensures that the nucleus obtains the materials it needs to synthesize RNA, while protecting the DNA from the hard environment outside the nucleus. i allowing RNA to leave the nucleus after it has been made.
“It’s a bit like an airplane hangar where the 747s can be repaired, and the door opens to let the 747 in, but there’s a person who can keep a single marble from coming out while the doors are open,” he says. André de Caltech. Hoelz, Professor of Chemistry and Biochemistry and Fellow of the Howard Hughes Medical Institute Faculty. For more than two decades, Hoelz has been studying and deciphering the structure of the NPC in relation to its function. Over the years, he has constantly erased his secrets, revealing them piece by piece.
The implications of this research are potentially enormous. NPC is not only central to cell operations, but is also involved in many diseases. Mutations in NPC are responsible for some incurable cancers, neurodegenerative and autoimmune diseases such as amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalopathy, and heart conditions such as atrial fibrillation and heart death. sudden early. In addition, many viruses, including the head of COVID-19, target and shut down the NPC over the course of its life cycles.
Now, in a couple of articles published in the magazine science, Hoelz and his research team describe two important advances: the determination of the structure of the outer face of the NPC and the elucidation of the mechanism by which special proteins act as a molecular glue to hold the NPC together.
A very small 3D puzzle
In their article entitled “Architecture of the cytoplasmic face of the nuclear pore”, Hoelz and his research team describe how they mapped the structure of the side of the NPC that looks outward from the core and 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 piece of the puzzle.
Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the first co-authors of the articles, says the process began with Escherichia coli bacteria (a strain of bacteria commonly used in laboratories) that were genetically modified to produce the proteins that make up the human NPC.
“If you walk into the lab, you can see that giant wall of jars in which cultures grow,” says Petrovic. “We express each protein individually E. coli cells, open those cells and chemically purify each protein component. “
Once this purification was completed, which may require up to 1,500 liters of bacterial culture to obtain enough material for a single experiment, the research team began to thoroughly test how the NPC pieces fit together.
George Mobbs, an associate chemistry senior postdoctoral researcher and another co-author of the paper, says the assembly took place “gradually”; instead of pouring all the proteins together into one test tube at a time, the researchers tested pairs of proteins to see which ones would fit together, like two pieces of a puzzle. If a matching pair was found, the researchers would test the two proteins now combined with a third protein until they found one that matched that pair, and then the resulting three-piece structure was tested with other proteins, and so activated. . Working through the proteins in this way ultimately produced the end result of his work: a 16-protein wedge that is repeated eight times, like slices of a 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, rather than reporting a series of incremental advances in fragments or portions based on partial, incomplete, or low resolution, “says Si Nie. , associate postdoctoral researcher in chemistry and also co-author of the paper. “We decided to wait patiently until we had acquired all the necessary data, reporting a large amount of new information.”
His work complemented research by Martin Beck of the Max Planck Institute for Biophysics in Frankfurt, Germany, whose team used cryoelectron tomography to generate a map that provided the outlines of a puzzle in which researchers had to place the pieces. To speed up the completion of the human NPC structure puzzle, Hoelz and Beck exchanged data more than two years ago and then independently constructed structures throughout the NPC. “Beck’s substantially improved map showed much more clearly where each piece of the NPC was to be placed, for which we determined the atomic structures, like a wooden frame that defines the edge of a puzzle.” , says Hoelz.
The experimentally determined structures of the NPC parts of the Hoelz group served to validate the modeling of the Beck group. “We placed the structures on the map independently, using different approaches, but the end results matched completely. It was very satisfying to see that,” says Petrovic.
“We built a framework on which a lot of experiments can now be done,” says Christopher Bley, a senior associate postdoctoral researcher in chemistry and also co-lead author. “We now have this composite structure and it allows and reports future experiments on NPC function, or even disease. There are many mutations in NPC that are associated with terrible disease, and knowing where they are in the structure and how together can help design the next set of experiments to try to answer questions about what these mutations do. ”
“This elegant spaghetti noodle arrangement”
In the other article, entitled “Architecture of the Nuclear Pore Link Scaffolding,” the research team describes how it determined the entire structure of what is known as the NPC’s link scaffold: the collection of proteins that help keep the NPC together. while providing the flexibility needed to open and close and adjust to adapt to the molecules that pass through it.
Hoelz compares the NPC to something made of Lego bricks that fit together without locking, and instead are joined by elastic bands that hold them in place even though they allow 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 / Caltech Laboratory
“I call these unstructured pieces of glue the ‘dark pore stuff,'” says Hoelz. “This elegant spaghetti noodle arrangement holds it all together.”
The process for characterizing the link scaffolding structure was very similar to the process used to characterize the other parts of the NPC. The team manufactured and purified large amounts of different types of binding and scaffolding 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 into the NPC intact.
To test their work, they introduced mutations in the genes encoding each of these binding proteins in a living cell. Because they knew how these mutations would change the chemical properties and shape of a specific binding protein, making it defective, they could predict what would happen to the structure of the cell’s NPCs when these defective proteins were introduced. If the NPCs in the cell were functionally and structurally defective in the way they expected, they knew they had the …