EMBL researchers have published two new studies involving the nuclear pore complex
From a tiny yeast specimen to a full-grown elephant, the defining feature of organisms known as eukaryotes is that they store their genetic information in the nucleus of their cells, surrounded by a protective double membrane. Embedded into the double membrane, nuclear pore complexes (NPCs) are large assemblies of about 1000 individual proteins called nucleoporins. The function of the NPC is that of a gateway between the nucleus and the rest of the cell. NPCs therefore play a substantial role in the exchange of large molecules and in regulating gene expression.
Many research groups at EMBL have studied the NPC. In 2016, scientists in the Beck group revealed the structure of the nuclear pore’s inner ring, and, in cooperation with the Ellenberg group, described its formation across the nucleus’s double membrane. Both the Beck group and the Ries group at EMBL have recently published new studies involving the NPC, addressing very different questions. The Beck group has uncovered a previously unknown formation process for the NPC, while the Ries group describes its use as a reference standard for super-resolution microscopy.
Observing disorder
“Our study describes the assembly of the nuclear core complex,” says Bernhard Hampoelz, a visiting researcher in the Beck group and lead author of the paper. “This is a very complicated process, because a thousand proteins need to assemble in the right order, in a relatively short time. Previously, we knew about two pathways that describe how this happens in tissue culture cells.” By studying the fruit fly Drosophila, the researchers were able to describe a third, alternative assembly mechanism that occurs during oogenesis: the formation of a fertile egg cell, or oocyte.
The two previously known pathways for NPC assembly take place at the nucleus, during different stages of the cell cycle. In the oocyte, NPCs are assembled outside the nuclear compartment and stored in membrane sheets called annulate lamellae. The oocyte prepares a stockpile of NPCs that are then inherited by the embryo and inserted into the nuclear envelope during its fast cell division cycles.
Andre Schwarz, a PhD student in the Beck group, explains that the study was done by creating a transgenic fly line that expressed fluorescent proteins in its NPCs, and observing NPC assembly using light and electron microscopy. “The NPC is deeply integrated into the double membrane, so it’s basically impossible to extract and purify,” says Schwarz. “We had to do everything in vivo. That meant dissecting Drosophila ovaries, culturing them in an imaging medium and then imaging them. Working in vivo is always more difficult.”
“But also a lot cooler,” Hampoelz adds with a grin. “You get to see all these processes in a living organism, in their natural context.”
Research in the Beck group has often focused on the structure of large molecular assemblies. “In our case, we go one step back, to the assembly process itself,” says Hampoelz. “There, everything still seems to be very disordered and very dynamic. We’re interested in seeing how such a complex machine is built out of a thousand proteins. It seems that, in oocytes, this happens from a very chaotic state. How exactly the assembly is regulated and the complexes are built up piece by piece in the oocyte is something we still don’t know much about.”
A new standard
The paper from the Ries group describes the use of NPCs as a reference standard for super-resolution microscopy. This was needed to benchmark the microscopy techniques that the group develops.
“The idea to use nuclear pores as a reference standard was quite straightforward,” says group leader Jonas Ries. “The Ellenberg group