Nuclear pores in their natural context

Study of the architecture of nuclear pores in yeast cells

September 02, 2020

Scientists from the Max Planck Institutes of Biophysics and Biochemistry and the European Molecular Biology Laboratory, led by Boris Pfander, Jan Kosinski und Martin Beck have studied the 3D structure of nuclear pores in budding yeast (Saccharomyces cerevisiae). Their findings reveal the architecture of the nuclear pore complex inside intact cells and increase our understanding of crucial processes of life.

Cryo-tomographic slice of a S. cerevisiae cell missing the protein Nup116 kept for 4 hours at the non-permissive temperature of 370C. Dotted rectangles mark the positions subjected to structural analysis; on the right cryo electron microscopy (cryoEM) maps (gray) with the respective fitted integrative models (colored).

Nuclear pores complexes (NPCs) are a highly complex assembly of proteins consisting of more than 500 individual proteins. Hundreds of them are embedded in the double membrane that surrounds and protects the cell’s nucleus. They act as a gateway that regulates the entry and exit of molecules. An important function of nuclear pores is to regulate the export of a molecule called mRNA (messenger ribonucleic acid) from the nucleus into the surrounding cell - the cytoplasm - where it delivers instructions for the assembly of proteins.

“We now appreciate better how the nuclear pore works in its native context, how it is maintained and recycled”, says group leader Martin Beck, who led the structural work. The researchers used a combination of cell biology, computational modelling, and cellular cryo-electron tomography: an imaging technique that is used to produce high-resolution 3D views of the molecular landscape inside a cell - This led to fundamental new insights: “We found out that the 3D configuration of the cytoplasmic ring accommodates the path of mRNA export”, says Matteo Allegretti, a postdoc in the Beck group and one of the first authors of the study.

Understanding the life cycle

The structure of the cytoplasmic ring also serves another function – it exposes a so-called AIM domain to the cytoplasm. “This domain interacts with proteins that facilitate the process by which nuclear pores are broken down by the cell and replaced with new ones – a process known as autophagic turnover”, says co-author Florian Wilfling (Wilfling was one of the scientists identifying this degradation pathway for nuclear pores and will continue his research studies at the Max Planck Institute of Biophysics as a Max Planck Research Group Leader in January 2021).

How exactly the architecture of nuclear pores facilitates the breakdown process - the so-called selective autophagy - and assembly of nuclear pores was largely unknown, but this study provides important first steps towards a better understanding of these mechanisms. “With knowledge coming from many different structures, we’re closer to understanding how nuclear pores assemble and how the pore evolved from the first cells with a nucleus up to now,” says group leader Jan Kosinski, who led the computational modelling.

To better understand the assembly of nuclear pores, the researchers grew a yeast strain missing a protein called nucleoporin 116 (Nup116), which plays an important role in the assembly process. The resulting structure was missing the cytoplasmic ring. The scientists conclude that these incomplete structures show intermediate states of nuclear pore assembly. Studying this process is important, as failures in the assembly of nuclear pores have been linked to neurodegenerative diseases.

The study yielded detailed structures of nuclear pores that scientists from other institutions can use in various ways, for example to study nuclear pore function, how molecules are transported into or out of the nucleus, or how viruses enter the nucleus. Many viruses, such as influenza and human immunodeficiency viruses (HIV), need to get their genetic information past the nuclear pore complex to infect a cell. “It also shows the scientific community that we need to shift scientific efforts towards the investigation of the structure-function relationship of macromolecules directly inside the cell,” says Matteo. Fundamental processes of life, such as nuclear transport and autophagy, can be understood by combining technologies like cryo-electron tomography with structural modelling, light microscopy, and biochemistry.

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