Understanding Nuclear Pore Complex biogenesis
The Nuclear Pore Complexes (NPCs) are elaborate nucleocytoplamic transport channels in the nuclear membrane of eukaryotic cells that are essential for cell viability. To maintain functional NPC population cells must be able to assemble new NPCs and eliminate dysfunctional ones. This is especially critical in cells with the intact nuclear membrane like neurones. In fact, many neurodegenerative disorders are associated with the NPC dysfunction. We use budding yeast as a model to investigate how cells manage NPC assembly in the intact nuclear membrane.
In eukaryotic cells genome is confined within membrane-enclosed cell nucleus requiring intense macromolecular communication across the nuclear border. It is estimated that every minute around 1kg of material crosses nuclear membrane in all cells in our body. This traffic occurs through 100 nm channels embedded into the nuclear membrane, the Nuclear Pore Complexes (NPC). The are ~ 2000-4000 of them in the human cell nucleus and ~ 100-200 in yeast.

The NPCs are among the most elaborate protein complexes in eukaryotic cells. Each structure is~ 100 MDa in size and consists of 500-1000 protein molecules - nucleoporins. There are around 30 different kinds of nucleoporins, each with own functional profile. The largest class, scaffold nucleoporins, form NPC walls and another big class - Phenyalanine-Glycine (FG) repeat nucleoporins - fill up the NPC transport channel. The FG segments are natively unfolded and work to prevent free NPC passage of any reasonably large macromolecules (>40 kDa), except for special Nuclear Transport Receptors (NTRs) that mediate nucleoytoplamic transport.

The interactions between NTRs, FG repeats, transported molecules and various regulatory factors establish nucleocytoplamic communication - the major NPC function. The NPCs have also other important roles in the cell including chromatin organisation, regulation of gene expression, DNA replication/repair or cell differentiation. Recently the NPC attracted a lot of attention being implicated in pathogenesis of many ageing-related disorders.

It is intriguing how new NPCs assemble. To make a new NPC cells must somehow produce a fusion pore in the membrane and to insert the multi-magadalton complex into it without perturbing the diffusion barrier by yet unclear mechanism. This is particularly non-trivial task in cells with the intact nuclear membrane like human neurones and could be the reason for accumulating NPC defects in the ageing brain. We are focusing on understanding the mechanisms of de novo NPC assembly using budding yeast, which is an extremely powerful genetic and biochemical model system. Although yeast is a unicellular organism, the yeast NPCs always stay in the intact nuclear membrane, keeping many parallels with the NPCs in cell types like human brain cells.
The blueprint of Nuclear Pore Complex assembly
How do NPCs assemble in live cells and how long does it take? To answer these questions we have recently developed KARMA - a technique that monitors incorporation kinetics of new proteins into protein complexes directly in live cells. In this project we aim to reveal details of the native NPC maturation process using KARMA, specifically focusing on the age-specific differences between NPCs, and molecular events accompanying different steps of the NPC assembly.

Our initial map of the NPC maturation generated with KARMA reveals many surprising details. For example, it takes ~ 1 hour to assemble a new NPC structure, which is on par with 90 minute generation time of yeast cells! For many nucleoporins the NPC incorporation takes only a few minutes but others need ~hour. Why the mauration times for some nucleoporins are so long is not yet clear. Our analysis also reveals that NPCs assemble in a specific ordered way where nucleoporins initially form sub-complexes that then co-assemble in a specific order into a mature NPC. It is especially curious that two nucleoprins called Mlp1 and Mlp2 assemble outstandingly late. Because of this, yeast have two co-existing populations of "old" and "new" NPCs that are compositionally different. What molecular events take place during the NPC maturation and what is the meaning of the age-specific differences in the NPC compotions? This project is aimed to answer such questions.
Nuclear Pore Complex assembly and nuncleocytoplamic transport
Among our main research interests is also understanding the role of nucleocytoplamic transport in the NPC assembly. We have found that genetic removal of FG repeats (responsible for nucleocytoplamic diffusion barrier) perturbs assembly of nucleoporins into the NPCs and in extreme cases completely inhibits NPC insertion into the nuclear membrane. The same FG repeats also physically intact with the scaffold nucleoporins which in turn can freely pass through the NPCs, pretty much like the NTRs. These and other intriguing connections between the NPC assembly and nucleocytoplamic transport is the subject of out ongoing research.
Contributions:
Project design: Evgeny Onishchenko (University of Bergen); Karsten Weis (ETHZ)
Mass spectrometry: Ludovic Gillet (ETHZ/Picotti lab); Evgeny Onishchenko (University of Bergen)
Microscopy & genetics: Evgeny Onishchenko (University of Bergen); Jonas Fischer (ETHZ/Weis lab); Carina Derrer (formerly ETHZ/Weis lab); Annemarie Kralt (formerly ETHZ/Weis lab)
Biochemistry: Evgeny Onishchenko (University of Bergen); Kasper Andersen (formerly MIT/Schwartz lab); Kevin Knockenhauer (formerly MIT/Schwartz lab)
Mathematical modelling: Elad Noor (Weizmann Institute/Milo lab)
In vitro transport assays: Jefrey Tang (formerly ETHZ/Weis lab)
Image analysis: Pascal Vallotton (Roche); Jonas Fischer (ETHZ/Weis lab)
EM: Evgeny Onishchenko (University of Bergen)
Visualisation: Jonas Fischer (ETHZ/Weis lab); Matthias Wojtynek (ETHZ/Weis lab); Olga Posukh (Institute of Molecular and Cell Biology, Novosibirsk)
Funding and research environment: Karsten Weis (ETHZ); Evgeny Onishchenko (University of Bergen); Thomas Schwartz (MIT)
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