Whatever you are doing, whether it is driving a car, going for a jog, or
even at your laziest, eating chips and watching TV on the couch, there is an
entire suite of molecular machinery inside each of your cells hard at work.
That machinery, far too small to see with the naked eye or even with many
microscopes, creates energy for the cell, manufactures its proteins, makes
copies of its DNA, and much more.
Among those pieces of machinery, and one of the most complex, is something
known as the nuclear pore complex (NPC). The NPC, which is made of more than
1,000 individual proteins, is an incredibly discriminating gatekeeper for
the cell's nucleus, the membrane-bound region inside a cell that holds that
cell's genetic material. Anything going in or out of the nucleus has to pass
through the NPC on its way.
The NPC's role as a gatekeeper of the nucleus means it is vital for the
operations of the cell. Within the nucleus, DNA, the cell's permanent
genetic code, is copied into RNA. That RNA is then carried out of the
nucleus so it can be used to manufacture the proteins the cell needs. The
NPC ensures the nucleus gets the materials it needs for synthesizing RNA,
while also protecting the DNA from the harsh environment outside the nucleus
and enabling the RNA to leave the nucleus after it has been made.
"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
Caltech's André Hoelz, professor of chemistry and biochemistry and a Faculty
Scholar of the Howard Hughes Medical Institute. 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 steadily chipped away at its
secrets, unraveling them piece by piece by piece by piece.
The implications of this research are potentially huge. Not only is the NPC
central to the operations of the cell, it is also involved in many diseases.
Mutations in the NPC are responsible for some incurable cancers, for
neurodegenerative and autoimmune diseases such as amyotrophic lateral
sclerosis (ALS) and acute necrotizing encephalopathy, and for heart
conditions including atrial fibrillation and early sudden cardiac death.
Additionally, many viruses, including the one responsible for COVID-19,
target and shutdown the NPC during the course of their lifecycles.
Now, in a pair of papers published in the journal Science, Hoelz and his
research team describe two important breakthroughs: the determination of the
structure of the outer face of the NPC and the elucidation of the mechanism
by which special proteins act like a molecular glue to hold the NPC
together.
A very tiny 3D jigsaw puzzle
In their paper titled "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 faces outward from the nucleus and into the cells'
cytoplasm. To do this, they had to solve the equivalent of a very tiny 3D
jigsaw 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 co-first authors of the papers, says the process began with
Escherichia coli bacteria (a strain of bacteria commonly used in labs) that
were 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 in which
cultures are growing," Petrovic says. "We express each individual protein in
E. coli cells, break those cells open, and chemically purify each protein
component."
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| A molecular model of the outside (cytoplasmic) face of the nuclear pore complex. Reprinted with permission from C.J. Bley et al., Science 376, eabm9129 (2022). Credit: Hoelz laboratory/Caltech |
Once that purification—which can require as much as 1,500 liters of
bacterial culture to get enough material for a single experiment—was
complete, the research team began to painstakingly test how the pieces of
the NPC fit together.
George Mobbs, a senior postdoctoral scholar research associate in chemistry
and another co–first author of the paper, says the assembly happened in a
"stepwise" fashion; rather than pouring all the proteins together into a
test tube at the same time, the researchers tested pairs of proteins to see
which ones would fit together, like two puzzle pieces. If a pair was found
that fit together, the researchers would then test the two now-combined
proteins against a third protein until they found one that fit with that
pair, and then the resulting three-piece structure was tested against other
proteins, and so on. Working their way through the proteins in this way
eventually produced the final result of their paper: 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, 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 also a 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."
Their work complemented research conducted by Martin Beck of the Max Planck
Institute of Biophysics in Frankfurt, Germany, whose team used cryo-electron
tomography to generate a map that provided the contours of a puzzle into
which the researchers had to place the pieces. To accelerate the completion
of the puzzle of the human NPC structure, Hoelz and Beck exchanged data more
than two years ago and then independently built structures of the entire
NPC. "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
says.
The experimentally determined structures of the NPC pieces from the Hoelz
group served to validate the modeling by the Beck group. "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 also co-first author. "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'
In the other paper, titled "Architecture of the linker-scaffold in the
nuclear pore," the research team describes how it determined the entire
structure of what is known as the NPC's linker-scaffold—the collection of
proteins that help hold the NPC together while also providing it with the
flexibility it needs to open and close and to adjust itself to fit the
molecules that pass through.
Hoelz likens the NPC to something built out of Lego bricks that fit together
without locking together and are instead lashed together by rubber bands
that keep them mostly in place while still allowing them to move around a
bit.
"I call these unstructured glue pieces the 'dark matter of the pore,'" Hoelz
says. "This elegant arrangement of spaghetti noodles holds everything
together."
The process for characterizing the structure of the linker-scaffold was much
the same as the process used to characterize the other parts of the NPC. The
team manufactured and purified large amounts of the many types 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 the intact NPC.
To check their work, they introduced mutations into the genes that code for
each of those linker proteins in a living cell. Since they knew how those
mutations would change the chemical properties and shape of a specific
linker protein, making it defective, they could predict what would happen to
the structure of the cell's NPCs 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.
"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 says.
The assembly of the NPC's outer face also helped solve a longtime mystery
about the nuclear envelope, the double membrane system that surrounds the
nucleus. Like the membrane of the cell within which the nucleus resides, the
nuclear membrane is not perfectly smooth. Rather, it is studded with
molecules called integral membrane proteins (IMPs) that serve 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 had been unclear how they actually traveled from one side to
the other. Indeed, because IMPs are stuck inside of the membrane, they
cannot just glide through the central transport channel of the NPC as do
free-floating molecules.
Once Hoelz's team understood the structure of the NPC's linker-scaffold,
they realized that it allows for the formation of little "gutters" around
its outside edge that allow the IMPs to slip past the NPC from one side of
the nuclear envelope to the other while always staying embedded in the
membrane itself.
"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 says.
Taken together, the findings of the two papers represent a leap forward in
scientists' understanding of how the human NPC is built and how it works.
The team's discoveries open the door for much more research. "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.
The papers describing the work appear in the June 10 issue of the journal
Science.
Reference:
Christopher J. Bley et al, Architecture of the cytoplasmic face of the
nuclear pore, Science (2022).
DOI: 10.1126/science.abm9129
Stefan Petrovic et al, Architecture of the linker-scaffold in the nuclear
pore, Science (2022).
DOI: 10.1126/science.abm9798