Biology subjects

van der Giessen, E.

Publications and source records attributed to van der Giessen, E..

7 recordsLinked to original sources

bioRxiv2025

Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

Nucleocytoplasmic transport occurs via nuclear pore complexes (NPCs), [~]40-60 nm wide pores lined with intrinsically disordered proteins that are rich in Phe-Gly motifs (FG-Nups) that form a selective barrier. Molecules larger than [~]50 kDa are increasingly blocked for transport unless they are bound to a nuclear transport receptor (NTR). How the amino acid sequence of FG-Nups contribute to this is not fully understood. Here, we present de novo designed artificial FG-Nups with a systematically varied FG-repeat spacing and charge-to-hydrophobicity ratio (C/H). Starting from a reference sequence termed NupY (with the average properties of natural yeast GLFG- Nups), we designed, synthesized, and experimentally tested a library of NupY variants using QCM-D experiments and phase separation assays. We find that the spacing between FG-motifs governs Kap95 absorption into the FG- Nup phase, while increasing C/H results in higher avidity for Kap95 due to an increased accessibility of FG-motifs. Molecular dynamics simulations of transport through NupY-coated pores show a reduced barrier function for noncohesive high-C/H-ratio variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between entropic repulsion and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.

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

Clearing-induced tissue shrinkage: Novel observation of a thickness size effect.

The use of clearing agents has provided new insights in various fields of medical research (developmental biology, neurology) by enabling examination of tissue architecture in 3D. One of the challenges is that clearing agents induce tissue shrinkage and the shrinkage rates reported in the literature are incoherent. Here, we report that the shrinkage for a widely-used clearing agent decreases significantly with increasing sample size, and report an analytical description.

biophysics↗

bioRxiv2020

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs.

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↗