Understanding Nuclear Pore Complex biogenesis
In eukaryotic cells genome is confined within membrane-enclosed cell nucleus requiring intense macromolecular communication across the nuclear border. It is estimated that in all cells in our body around 1kg of macromolecules cross the nuclear membrane every minute. This traffic is accomplished by multiprotein Nuclear Pore Complex (NPC) channels embedded in the nuclear membrane. The are ~ 2000 NPCs in the human cell nucleus and ~ 150 in the yeast nucleus.
The NPCs are gigantic mucltiprotein channels measuring around 100 nm in diameter and consisting of 500-1000 nucleoporin proteins depending on the species (see image on the side). Some or the numerous nucleoporins form NPC scaffold while and others fill up the NPC transport channel thanks to special naively disordered Phenyalanine-Glycine (FG) repeat segments. Wheres FG segments prevent free diffusion of macromolecules above ~ 40 kDa, specific Nuclear Transport Receptors (NTRs) mediate directional nucleoytoplamic transport of macromoleculer cargos that could be as large as the viral particles comparable in dimensions to the NPC itself.
Structure of the Nuclear Pore Complex
Adapted from: Bley et.al., 2022 (Andre Hoelz lab)
To make a new NPC cells must seamlessly insert the gigantic complex into the nuclear membrane and if the NPC is out of order they must be somehow eliminated or repaired to maintain them properly functional. It is especially intriguing how this is accomplished in cells that never divide (like neurones) and therefore must control their NPCs while keeping the nuclear membrane intact. In fact NPC dysfunction in the non-proliferating cell types recently attracted attention as the hotspot of age-related disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's and Parkinson's diseases and various myodystrophies.
Our group is interested in understanding cellular control over the events of the NPC lifecycle and the challenges posed by the intact nuclear membrane. We address these topics by investigating NPC lifecycle from assembly to elimination in budding yeast, which is an excellent 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 with non-dividing human cells. Thanks to 90 minute generation time, any genetic manipulations in yeast take only couple of days thus allowing to test various hypotheses.
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. In this project we are focusing on understanding the mechanisms of de novo NPC assembly and its connections with the nucleocytoplamic transport function of the NPC.
One intriguing connection we have found is that elimination of the barrier-forming FG repeats perturbs NPC assembly and in extreme cases completely blocks NPC insertion into the intact nuclear membrane (see slides below). It is most striking these FG repeats physically intact with the scaffold nucleoporins. These intriguing connections suggest that NPC assembly and nucleocytoplamic transport are functionally which is the subject of our ongoing research.
Nuclear Pore Complex assembly and nucleocytoplasmic transport
Nuclear Pores Complexes (NPC) form channels in the nuclear membrane (grey) connecting cell nucleus with the cytoplasm. The NPC channels are shaped by scaffold nucleoporins (blue) and filled with FG repeats (green wires) that block free passage of macromolecules. The only exception is nuclear transport receptors (NTRs) that can freely pass through the FG repeats and carry different macromolecules across.
It is also not entirely understood how cells regulate the NPC assembly process. We have found several intriguing connections between NPC-mediated transport and NPC assembly. For example, scaffold nulceoporins like Nic96 (left) have similar properties as NTRs (Importin-beta, middle) and can get inside cell nucleus through NPCs. For comparison, regular proteins like three linked GFP molecules (right) cannot get inside cell nucleus in spite of their smaller size.
These and other experimental evidence point to a deep connection between nucleocytoplamic transport and NPC assembly. FG repeats likely govern important control steps in the NPC assembly to ensure a complete diffusion barrier in the newly formed NPCs. The mechanism of such control is a topic of the ongoing research in the lab.
Nuclear Pores Complexes (NPC) form channels in the nuclear membrane (grey) connecting cell nucleus with the cytoplasm. The NPC channels are shaped by scaffold nucleoporins (blue) and filled with FG repeats (green wires) that block free passage of macromolecules. The only exception is nuclear transport receptors (NTRs) that can freely pass through the FG repeats and carry different macromolecules across.
The blueprint of Nuclear Pore Complex assembly
What is the order NPC biogenesis and how long does it take for the cell to make a new NPC? 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 current map of the NPC maturation generated with KARMA reveals many surprising details (see slides below). 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. While our findings began to uncover a mysteries behind the assembly and compositional diversity of the NPCs may questions remain to be answered. 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.
It is still not known how cells assemble NPCs into the nuclear membrane. To tackle this in a systematic way we have developed KARMA - a method that analyses assembly of protein complexes in live cells by pulse-labelling the proteins with heavy isotope amino acids (SILAC). The protein complexes (e.g. NPCs) are then isolated via affinity tags and labelling kinetics of the complex components is measured by quantitative mass spectrometry (AP-MS).
Our KARMA-based analysis revels several interesting patterns. First, all newly-made nucleoporins initially co-assemble into distinct anatomical parts of the NPC (assembly groups) that typically occurs on a minute timescale.
Because of the late Mlp's assembly, the yeast cells have two NPC populations: the "young" NPCs that have no Mlps (left) and the "old" ones that have them (right). In our group we are using KARMA-based approaches to better understand the purpose of these age differences and what other molecular events happen during the NPC assembly.
It is still not known how cells assemble NPCs into the nuclear membrane. To tackle this in a systematic way we have developed KARMA - a method that analyses assembly of protein complexes in live cells by pulse-labelling the proteins with heavy isotope amino acids (SILAC). The protein complexes (e.g. NPCs) are then isolated via affinity tags and labelling kinetics of the complex components is measured by quantitative mass spectrometry (AP-MS).
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)
High-throughput analysis of protein dynamics
Protein dynamics can be thought of as a chain of events that cellular proteins undergo from the moment of biosynthesis and until elimination. Examples of such events are synthesis on ribosomes, protein folding, interaction with other proteins, proteasomal degradation etc... In spite of the paramount importance, protein dynamics is a highly understudied topic. For example, the assembly pathways (the way proteins interact with one another to form complexes) are known only for a handful of ~4000 human protein complexes. This project focuses on the development of tools to explore dynamics of cellular protein complexes.
Our approach to tackle protein dynamics is based on the idea that biogenesis of protein complexes can be seen as a flow of metabolic reactions except that the "metabolites" are not typical small molecules but cellular proteins and their assemblies. Based on this idea we have recently developed an approach entitled kinetic analysis of incorporation rates in macromolecular assemblies (KARMA) that utilises a combination of isotope metabolic labelling and quantitative mass spectrometry to dissect in vivo dynamics of protein complexes. This project amis at development of the analytical techniques to address protein dynamics of various cellular protein complexes, host-pathogen interactions or other kinds of biomolecules.
Cellular proteins form thousands of different complexes but almost nothing is known about how they assemble or change in time. To study these processes we have developed Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA) - a method that relies on metabolic labelling to lean about the timing and order of the protein complexes assembly. In KARMA the complex assembly is treated akin to metabolic reactions, each taking some time. This creates a delay between the initial metabolite exposure (e.g. feeding cells with isotope labeled amino acids) and the time it incorporates into the complexes. This delay can be experimentally measured by isolating the protein complex through different complex components as affinity baits and analysing the metabolic label content by mass spectrometry. The complex constituent's labelling is then matched with a theoretical description - Kinetic State Model - to find the maturation time for every complex component, fraction of proteins in the assembly intermediates and and other important parameters. We apply this strategy to study various assembly processes.
Contributions
Project design: Evgeny Onishchenko (University of Bergen)
Biochemistry: Evgeny Onishchenko (University of Bergen)
Mass spectrometry: Ludovic Gillet (ETHZ/Picotti lab); Evgeny Onishchenko (University of Bergen)
Mathematical modelling: Elad Noor (Weizmann Institute/Milo lab)
Microscopy & genetics: Jonas Fischer (ETHZ/Weis lab)
Image analysis: Pascal Vallotton (Roche AG); Jonas Fischer (ETHZ/Weis lab)
Visualisation: Jonas Fisher (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)
Dynamic profiling of proteostasis network
Quality of cellular proteins is controlled by a sophisticated machinery - proteostasis network - including folding chaperones, disaggregates, autophagy machinery, proteasome and numerous other factors. It is estimated that more than 10% of cellular proteome is devoted to the protein quality control. What is the functional specialisation of the PN elements? At which stage of the protein's lifecycle the PN interrogates its clients? We are seeking answers to these questions by profiling in a time-resolved manner the interactions of PN with the cellular proteome using quantitative mass-spectrometry and mathematical models based on proteomic data.