Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.

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Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903764116 ◽

2019 ◽

Vol 116 (29) ◽

pp. 14606-14613 ◽

Cited By ~ 8

Author(s):

Pascal Vallotton ◽

Sasikumar Rajoo ◽

Matthias Wojtynek ◽

Evgeny Onischenko ◽

Annemarie Kralt ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nucleocytoplasmic Transport ◽

Live Cells ◽

Nuclear Pore ◽

Intrinsically Disordered ◽

Intensity Measurements ◽

Composition And Structure ◽

Associated Proteins ◽

Multiple Copies

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

10.1101/2020.10.09.334029 ◽

2020 ◽

Author(s):

Tae Yeon Yoo ◽

Timothy J Mitchison

Keyword(s):

Nuclear Import ◽

Nuclear Transport ◽

Super Resolution ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Import And Export ◽

Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

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In vivo analysis of human nucleoporin repeat domain interactions

Molecular Biology of the Cell ◽

10.1091/mbc.e12-08-0585 ◽

2013 ◽

Vol 24 (8) ◽

pp. 1222-1231 ◽

Cited By ~ 25

Author(s):

Songli Xu ◽

Maureen A. Powers

Keyword(s):

Fluorescent Protein ◽

Live Cells ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nucleocytoplasmic Trafficking ◽

Repeat Domain ◽

Domain Interactions ◽

In Vivo Analysis ◽

Green Fluorescent

The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier.

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Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport

The Journal of Cell Biology ◽

10.1083/jcb.201201087 ◽

2012 ◽

Vol 198 (3) ◽

pp. 343-355 ◽

Cited By ~ 29

Author(s):

Gero Steinberg ◽

Martin Schuster ◽

Ulrike Theisen ◽

Sreedhar Kilaru ◽

Andrew Forge ◽

...

Keyword(s):

Nuclear Lamina ◽

Ustilago Maydis ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nuclear Pores ◽

Gfp Reporter ◽

Import And Export ◽

Reporter Proteins ◽

Pore Complexes

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ∼1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.

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Promoter-proximal poly(A) sites are processed efficiently, but the RNA products are unstable in the nucleus.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2127 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2127-2135 ◽

Cited By ~ 5

Author(s):

J M Scott ◽

M J Imperiale

Keyword(s):

Steady State ◽

Nucleocytoplasmic Transport ◽

Proximity Effects ◽

Expression Vectors ◽

Genome Replication ◽

Intact Cells ◽

A Site ◽

Hiv 1

The presence of two polyadenylation signals in the primary transcript of the human immunodeficiency virus type 1 (HIV-1) provirus leads to a requirement for regulation of 3'-end processing. To ensure that viral genome replication and gene expression occur, polyadenylation must occur at the poly(A) site transcribed from the 3' long terminal repeat (LTR) but not the 5' LTR. Models that have been proposed to explain this regulation include (i) inhibition of the 5' site as a result of proximity to the promoter and (ii) enhancement of the 3' site by U3 sequences that are transcribed upstream of only the 3' poly(A) site. In previous studies designed to investigate these models, a reduction in the levels of steady-state RNA was observed when the HIV-1 poly(A) site was placed within 500 nucleotides of the cap site. Although these findings were interpreted to be the result of promoter proximity effects on 3'-end processing, in vitro studies demonstrated that the HIV-1 poly(A) site was equally functional in promoter-proximal and promoter-distal positions. These results led to the hypothesis that, in vivo, the poly(A) site is fully active at this close distance but the short transcripts produced are highly unstable in the nucleus and undergo rapid degradation, precluding their appearance as abundant mRNAs in the steady-state pool. To investigate the biogenesis of these short RNAs in vivo, experiments were performed to examine directly the nuclear processing rates of the HIV-1 poly(A) site in intact cells. By using recombinant adenoviruses as expression vectors, it is now demonstrated conclusively that the HIV-1 poly(A) site is indeed processed at equivalent levels independent of its distance from the promoter. Although transcripts containing the promoter-proximal poly(A) site are processed efficiently, most undergo degradation in the nucleus instead of nucleocytoplasmic transport.

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Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.955 ◽

1989 ◽

Vol 109 (3) ◽

pp. 955-970 ◽

Cited By ~ 126

Author(s):

C W Akey

Keyword(s):

Electron Microscopy ◽

Mirror Symmetry ◽

Nuclear Lamina ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Effective Diameter ◽

Nuclear Pore Complexes ◽

Cryo Electron Microscopy ◽

Symmetrical Configuration

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

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The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200407156 ◽

2004 ◽

Vol 167 (4) ◽

pp. 583-590 ◽

Cited By ~ 56

Author(s):

Bryan Zeitler ◽

Karsten Weis

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Pore Complex ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleocytoplasmic Trafficking ◽

Transport Pathways ◽

Pore Complexes ◽

Biased Distribution

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.

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Nup62 is recruited to pathological condensates and promotes TDP-43 insolubility in C9orf72 and sporadic ALS/FTLD.

10.21203/rs.3.rs-144654/v1 ◽

2021 ◽

Author(s):

Amanda Gleixner ◽

Brandie Morris Verdone ◽

Charlton Otte ◽

Nandini Ramesh ◽

Jenna Gale ◽

...

Keyword(s):

Phase Transitions ◽

Nucleocytoplasmic Transport ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nucleocytoplasmic Trafficking ◽

Mechanisms Of Toxicity ◽

Sporadic Als

Abstract Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) share clinical, neuropathological, and genetic features. This includes common genetic disease-causing mutations such as the expanded G4C2 repeat in the C9orf72 gene (C9-ALS/FTLD) and cytoplasmic and insoluble protein depositions of the TDP-43 in degenerating regions of the brain and spinal cord. Proposed mechanisms of toxicity in C9-ALS/FTLD are the production of repeat expansion transcripts and their dipeptide repeat proteins (DPRs) products which are hypothesized to drive nucleocytoplasmic transport defects. The nuclear pore complex (NPC) regulates nucleocytoplasmic trafficking by creating a selectivity and permeability barrier comprised of phenylalanine glycine nucleoporins (FG nups). However, the relationship between FG nups and TDP-43 pathology remains elusive. Here, we define two mechanisms through which TDP-43 promotes Nup62 nuclear depletion and cytoplasmic in C9-ALS/FTLD and sALS/FTLD. In C9-ALS/FTLD, poly-GR initiates the formation of TDP-43 containing stress granules (SGs) that trigger the nuclear loss and recruitment of Nup62 in vitro and in vivo. When colocalized, cytoplasmic TDP-43:Nup62 assemblies mature into insoluble inclusions through an interaction within the TDP-43 nuclear localization sequence (NLS) suggesting Nup62 promotes deleterious phase transitions. Absent of poly-GR, aberrant TDP-43 phase transitions in the cytoplasm recruits and mislocalizes Nup62 into pathological inclusions. The result of these cytoplasmic Nup62 and TDP-43 interactions are pathological and insoluble TDP-43:Nup62 assemblies that are observed in C9-ALS/FTLD and sALS/FTLD CNS tissue.

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Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1

Journal of Cell Science ◽

10.1242/jcs.112.14.2369 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2369-2380 ◽

Cited By ~ 1

Author(s):

O. Rosorius ◽

B. Reichart ◽

F. Kratzer ◽

P. Heger ◽

M.C. Dabauvalle ◽

...

Keyword(s):

Nuclear Export ◽

Mammalian Cells ◽

Direct Interaction ◽

Nucleocytoplasmic Transport ◽

Cellular Protein ◽

Initiation Factor ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Export Receptor

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.

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