Context matters. This is 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 October 13 in the journal Nature suggests that this practice can drastically modify the components in question. Researchers have developed a method to study a large donut-shaped structure called a nuclear pore complex (NPC) directly inside cells. Their results revealed that the pore had larger dimensions than previously thought, highlighting the importance of analyzing complex molecules in their native environment.
“We have shown that the cellular environment has a significant impact on large structures like NPC, which we did not expect when we first started,” says Thomas Schwartz, Boris Magasanik professor of biology and co-lead author of the study. . “Scientists have generally believed that large molecules are stable enough to maintain their fundamental properties both inside and outside a cell, but our findings reverse this hypothesis.”
In eukaryotes like humans and animals, most of a cell’s DNA is stored in a rounded structure called the nucleus. This organelle is protected 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 get in and out of the nucleus in order to facilitate important processes, including gene expression. This is 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 postdoctoral fellow Anthony Schuller, compares NPCs to the gates of a sports stadium. “If you want to access the game inside, you have to show your ticket and go through one of these doors,” he explains.
The NPC might be tiny by human standards, but it’s one of the largest structures in the cell. It is made up of around 500 proteins, which made its structure difficult to analyze. Traditionally, scientists have broken it down 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 does not is available only recently.
In collaboration with researchers at the University of Zurich, Schuller and Schwartz used two cutting-edge approaches to solve pore structure: cryo-focused ion beam grinding (cryo-FIB) and cryo-electron tomography (cryo- AND).
An entire cell is too thick to be seen under an electron microscope. But the researchers cut frozen colon cells into thin layers using cryo-FIB equipment housed at the Center for Automated Cryogenic Electron Microscopy at MIT.nano and the Peterson (1957) Nanotechnology Materials Core Facility at the Koch Institute. In doing so, the team captured cross sections of cells that included NPCs, rather than just looking at NPCs in isolation.
“The amazing thing about this approach is that we barely manipulated the cell,” says Schwartz. “We have not disturbed the internal structure of the cell. It is the revolution.”
What the researchers saw when looking at their microscopy images was quite different from existing descriptions of the NPC. They were surprised to find that the innermost ring structure, which forms the central channel of the pore, is much wider than previously thought. When left in its natural environment, the pore opens up to 57nm, resulting in a 75% increase in volume over previous estimates. The team was also able to take a closer look at how the different components of the NPC work together to define the pore dimensions and the overall architecture.
“We have shown that the cellular environment has an impact on the structure of NPCs, but now we have to understand how and why,” says Schuller. 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 article is a good illustration of how technical advances, in this case cryoelectron tomography on human cells crushed by cryo-focused ion beams, provide a new picture of cellular structures,” says Wolfram Antonin, professor of biochemistry at the RWTH University of Aachen in Germany which did not participate in the study. The fact that the diameter of the central transport channel of the NPC is larger than previously thought suggests that the pore could have impressive structural flexibility. “This can be important for the cell to adapt to increased transport demands,” explains Antonin.
Next, Schuller and Schwartz hope to understand how the size of the pore affects the molecules that can pass through it. For example, scientists only recently determined that the pore was large enough to allow intact viruses like HIV to enter the nucleus. The same principle applies to medical treatments: only drugs of the appropriate size and with specific properties will be able to access the DNA of the cell.
Schwartz is especially curious as to whether all NPCs are created equal, or if their structure differs between species or cell types.
“We have always manipulated cells and taken individual components out of their native context,” he says. “Now we know that this method can have much bigger consequences than we thought.”