Organelle-specific targeting of polymersomes into the cell nucleus

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

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Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export

Journal of Cell Science ◽

10.1242/jcs.111.2.223 ◽

1998 ◽

Vol 111 (2) ◽

pp. 223-236 ◽

Cited By ~ 4

Author(s):

E. Kiseleva ◽

M.W. Goldberg ◽

T.D. Allen ◽

C.W. Akey

Keyword(s):

Nuclear Export ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Internal Ring ◽

Balbiani Ring ◽

Transmission Electron ◽

The Individual ◽

Pore Complexes ◽

Small Channels

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

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Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide

Nature Communications ◽

10.1038/s41467-021-24292-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sheung Chun Ng ◽

Thomas Güttler ◽

Dirk Görlich

Keyword(s):

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nuclear Pore Complexes ◽

Cell Nuclei ◽

Cargo Transport ◽

Fast Transport ◽

Import And Export ◽

Pore Complexes ◽

Fundamental Requirement

AbstractThe permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with ‘DNA-specific’ DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.

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SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

The Journal of Cell Biology ◽

10.1083/jcb.140.3.499 ◽

1998 ◽

Vol 140 (3) ◽

pp. 499-509 ◽

Cited By ~ 316

Author(s):

Michael J. Matunis ◽

Jian Wu ◽

Günter Blobel

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Gtpase Activating Protein ◽

Terminal Domain ◽

Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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In Vivo Dynamics of Nuclear Pore Complexes in Yeast

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1185 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1185-1199 ◽

Cited By ~ 82

Author(s):

Mirella Bucci ◽

Susan R. Wente

Keyword(s):

Nuclear Envelope ◽

Recipient Cell ◽

Nucleocytoplasmic Transport ◽

Yeast Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Central Location ◽

Nuclear Membranes ◽

Mutant Strains ◽

Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

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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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Nuclear Pore Proteins in Regulation of Chromatin State

Cells ◽

10.3390/cells8111414 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1414 ◽

Cited By ~ 7

Author(s):

Terra M. Kuhn ◽

Maya Capelson

Keyword(s):

Nucleosome Occupancy ◽

Regulation Of Gene Expression ◽

Nucleocytoplasmic Transport ◽

Chromatin State ◽

Nuclear Pore ◽

Regulation Of Transcription ◽

Nuclear Pore Complexes ◽

Functional Connection ◽

Histone Modifiers ◽

Pore Complexes

Nuclear pore complexes (NPCs) are canonically known to regulate nucleocytoplasmic transport. However, research efforts over the last decade have demonstrated that NPCs and their constituent nucleoporins (Nups) also interact with the genome and perform important roles in regulation of gene expression. It has become increasingly clear that many Nups execute these roles specifically through regulation of chromatin state, whether through interactions with histone modifiers and downstream changes in post-translational histone modifications, or through relationships with chromatin-remodeling proteins that can result in physical changes in nucleosome occupancy and chromatin compaction. This review focuses on these findings, highlighting the functional connection between NPCs/Nups and regulation of chromatin structure, and how this connection can manifest in regulation of transcription.

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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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Targeting and function in mRNA export of nuclear pore complex protein Nup153.

The Journal of Cell Biology ◽

10.1083/jcb.134.5.1141 ◽

1996 ◽

Vol 134 (5) ◽

pp. 1141-1156 ◽

Cited By ~ 136

Author(s):

R Bastos ◽

A Lin ◽

M Enarson ◽

B Burke

Keyword(s):

Nuclear Pore Complex ◽

Protein Import ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Terminal Domain ◽

General Decline ◽

Complex Protein ◽

Pore Complexes ◽

And Function

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

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Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0244 ◽

2016 ◽

Vol 27 (17) ◽

pp. 2742-2756 ◽

Cited By ~ 14

Author(s):

Biplab Paul ◽

Ben Montpetit

Keyword(s):

Single Molecule ◽

Rna Processing ◽

Nuclear Export ◽

Essential Gene ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Eukaryotic Gene ◽

Transcription Sites ◽

Pore Complexes

Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

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A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.939 ◽

1995 ◽

Vol 129 (4) ◽

pp. 939-955 ◽

Cited By ~ 119

Author(s):

L C Gorsch ◽

T C Dockendorff ◽

C N Cole

Keyword(s):

Messenger Rna ◽

Mrna Export ◽

Temperature Shift ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nuclear Pore Complexes ◽

Reporter Protein ◽

Direct Role ◽

Pore Complexes

In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.

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