Cellular environments shape molecular architecture
Researchers glean a more complete picture of a structure called the
nuclear pore complex by studying it directly inside cells.

Date:
October 15, 2021
Source:
Massachusetts Institute of Technology Department of Biology
Summary:
An important cellular structure called the nuclear pore complex
(NPC) has larger dimensions than previously thought. A research
team made this discovery using cryo-focused ion beam (cryo-FIB)
milling and cryo- electron tomography (cryo-ET) -- which allowed
them to analyze the NPC directly inside cells.



FULL STORY ========================================================================== Context matters. It's true for many facets of life, including the tiny molecular machines that perform vital functions inside our cells.


========================================================================== Scientists often purify cellular components, such as proteins or
organelles, in order to examine them individually. However, a new study published on Oct. 13 in the journal Nature suggests that this practice
can drastically alter the components in question. The researchers devised
a method to study a large, donut-shaped structure called the nuclear
pore complex (NPC) directly inside cells. Their results revealed that
the pore had larger dimensions than previously thought, emphasizing the importance of analyzing complex molecules in their native environments.

"We've shown that the cellular environment has a significant impact
on large structures like the NPC, which was something we weren't
expecting when we started," says Thomas Schwartz, the Boris Magasanik
Professor of Biology and the study's co-senior author. "Scientists have generally thought that large molecules are stable enough to maintain
their fundamental properties both inside and outside a cell, but our
findings turn that assumption on its head." In eukaryotes like humans and animals, most of a cell's DNA is stored in a rounded structure called the nucleus. This organelle is shielded by the nuclear envelope, a protective barrier that separates the genetic material in the nucleus from the thick
fluid filling the rest of the cell. But molecules still need a way to come
in and out of the nucleus in order to facilitate important processes,
including gene expression. That's where the NPC comes in. Hundreds -
- sometimes thousands -- of these pores are embedded in the nuclear
envelope, creating gateways that allow certain molecules to pass.

The study's first author, former postdoc Anthony Schuller, compares NPCs
to gates at a sports stadium. "If you want to access the game inside, you
have to show your ticket and go through one of these gates," he explains.

The NPC may be tiny by human standards, but it's one of the largest
structures in the cell. It's comprised of roughly 500 proteins, which
has made its structure challenging to parse. Traditionally, scientists
have broken it up into individual components to study it piecemeal
using a method called X-ray crystallography. According to Schwartz,
the technology required to analyze the NPC in a more natural environment
has only recently become available.



========================================================================== Together with researchers from the University of Zurich, Schuller
and Schwartz employed two cutting-edge approaches to solve the pore's structure: cryo- focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET).

An entire cell is too thick to look at under an electron microscope. But
the researchers sliced frozen colon cells into thin layers using
the cryo-FIB equipment housed at MIT.nano's Center for Automated
Cryogenic Electron Microscopy and the Koch Institute's Peterson (1957) Nanotechnology Materials Core Facility. In doing so, the team captured cross-sections of the cells that included NPCs, rather than simply
looking at the NPCs in isolation.

"The amazing thing about this approach is that we've barely manipulated
the cell at all," Schwartz says. "We haven't perturbed the cell's
internal structure. That's the revolution." What the researchers saw
when they looked at their microscopy images was quite different from
existing descriptions of the NPC. They were surprised to find that
the inner most ring structure, which forms the pore's central channel,
is much wider than previously thought. When it's left in its natural environment, the pore opens up to 57 nm -- resulting in a 75% increase in volume compared to previous estimates. The team was also able to take a
closer look at how the NPC's various components work together to define
the pore's dimensions and overall architecture.

"We've shown that the cellular environment impacts NPC structure, but now
we have to figure out how and why," Schuller says. Not all proteins can
be purified, he adds, so the combination of cryo-ET and cryo-FIB will also
be useful for examining a variety of other cellular components. "This dual approach unlocks everything." "The paper nicely illustrates how technical advances, in this case cryo- electron tomography on cryo-focused ion beam milled human cells, provide a fresh picture of cellular structures," says Wolfram Antonin, a professor of biochemistry at RWTH Aachen University
in Germany who was not involved in the study. The fact that the diameter
of the NPC's central transport channel is larger than previously thought
hints that the pore could have impressive structural flexibility. "That
may be important for the cell to adapt to increased transport demands,"
Antonin explains.

Next, Schuller and Schwartz hope to understand how the size of the
pore affects which molecules can pass through. For instance, scientists
only recently determined that the pore was big enough to allow intact
viruses like HIV into the nucleus. The same principle applies to medical treatments: only appropriately-sized drugs with specific properties will
be able to access the cell's DNA.

Schwartz is especially curious to know whether all NPCs are created equal,
or if their structure differs between species or cell types.

"We've always manipulated cells and taken the individual
components out of their native context," he says. "Now we know
this method may have much bigger consequences than we thought." ========================================================================== Story Source: Materials provided by Massachusetts_Institute_of_Technology_Department_of Biology. Original
written by Raleigh McElvery. Note: Content may be edited for style
and length.


========================================================================== Journal Reference:
1. Anthony P. Schuller, Matthias Wojtynek, David Mankus, Meltem Tatli,
Rafael Kronenberg-Tenga, Saroj G. Regmi, Phat V. Dip, Abigail K. R.

Lytton-Jean, Edward J. Brignole, Mary Dasso, Karsten Weis,
Ohad Medalia, Thomas U. Schwartz. The cellular environment
shapes the nuclear pore complex architecture. Nature, 2021; DOI:
10.1038/s41586-021-03985-3 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/10/211015111207.htm

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