Biology subjects

Onck, P.

Publications and source records attributed to Onck, P..

6 recordsLinked to original sources

bioRxiv2023

C9orf72 polyPR directly binds to various nuclear transport components

The disruption of nucleocytoplasmic transport (NCT) is an important mechanism in neurodegenerative diseases. In the case of C9orf72-ALS, trafficking of macromolecules through the nuclear pore complex (NPC) might get frustrated by the binding of C9orf72-translated arginine-containing dipeptide repeat proteins (R-DPRs) to the Kap{beta} family of nuclear transport receptors. Beside Kap{beta}s, several other types of transport components have been linked to NCT impairments in R-DPRs expressed cells, but the molecular origin of these observations has not been clarified. Here, we adopt a coarse-grained molecular dynamics model at amino-acid resolution to study the direct interaction between polyPR, the most toxic DPR, and various nuclear transport components to elucidate the binding mechanisms and provide a complete picture of potential polyPR-mediated NCT defects. We found polyPR to directly bind to several isoforms of the Imp family, CAS (the specific exporter of Imp) and RanGAP. We observe no binding between polyPR and Ran. Longer polyPRs at lower salt concentrations also make contact with RanGEF and NTF2. Analyzing the polyPR contact sites on the transport components reveals that polyPR potentially interferes with RanGTP/RanGDP binding, with nuclear localization signal (NLS)-containing cargoes (cargo-NLS) binding to Imp, with cargo-NLS release from Imp, and with Imp export from the nucleus. The abundance of polyPR binding sites on multiple transport components combined with the inherent polyPR length dependence makes direct polyPR interference of NCT a potential mechanistic pathway of C9orf72 toxicity.

biophysics↗

bioRxiv2023

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 {+/-} 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular-dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

biophysics↗

bioRxiv2022

Liquid-liquid phase separation of intrinsically disordered FG-Nups is driven by highly-dynamic hydrophobic FG-motifs

The intrinsically disordered FG-Nups in the NPC central channel can undergo liquid-liquid phase sepration (LLPS) into liquid condensates that display NPC-like permeability barrier properties. Here we present LLPS characteristics of each of the disordered FG-Nups of the yeast NPC. Using molecular dynamics simulations at amino acid resolution, FG-Nup condensates are studied and the main physicochemical driving forces for FG-Nup LLPS are identified. We show that FG-motifs that are predominantly present in the disordered domain of FG-Nups act as highly-dynamic hydrophobic stickers that are essential for the formation of stable liquid-like condensates. Next to that, we study LLPS of an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple types of FG-Nups. We find that the LLPS of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed LLPS behavior, we categorize the different FG-Nups of the yeast NPC into two classes: The GLFG-Nups located in the central channel of the NPC phase separate into liquid-like condensates, forming a high-density cohesive barrier that can exclude inert particles. The FG-Nups at the entry and exit of the NPC channel, containing no GLFG-motifs, do not phase separate and possibly form a repulsive barrier by entropically excluding inert particles.

biophysics↗

bioRxiv2021

Transport receptor occupancy in Nuclear Pore Complex mimics

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess Kap-centric NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

biophysics↗

bioRxiv2020

Phase separation of toxic dipeptide repeat proteins related to C9orf72 ALS/FTD

The expansion mutation in the C9orf72 gene is the most common known genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation can produce five dipeptide repeat proteins (DPRs), of which three are known to be toxic: poly-PR, poly-GR, and poly-GA. The toxicity of poly-GA is attributed to its aggregation in the cytoplasm, while for poly-PR and poly-GR several toxicity pathways have been proposed. The toxicity of the DPRs has been shown to depend on their length, but the underlying molecular mechanism of this length dependence is not well understood. To address this, a one-bead-per-amino-acid (1BPA) coarse-grained molecular dynamics model is used to study the single-molecule and phase separation properties of the DPRs. We find a strong dependence of the phase separation behavior on both DPR length and concentration, with longer DPRs having a higher propensity to phase separate and form condensed phases with higher concentrations. The critical lengths required for phase separation (25 for poly-PR and 50 for poly-GA) are comparable to the toxicity threshold limit of 30 repeats found for the expansion mutation in patient cells, suggesting that phase separation could play an important role in DPR toxicity.

biophysics↗

bioRxiv2018

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC function is compromised in ageing mitotic cells. By imaging of single yeast cells during ageing, we show that the abundance of several NPC components and NPC assembly factors decreases while signs of misassembled NPCs appear. Consequently, nuclear permeability decreases, resulting in decreased dynamics of transcription factor shuttling and increased nuclear compartmentalisation. In support that declining NPC quality control is important in mitotic ageing, we find that the transport kinetics observed in ageing is mimicked in an NPC assembly mutant. Additionally, the single cell life histories reveal that cells that better maintain NPC function are longer lived. We conclude that assembly and quality control of NPCs are major challenges for ageing mitotic cells.

cell biology↗