scholarly journals Investigating molecular crowding within nuclear pores using polarization-PALM

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Guo Fu ◽  
Li-Chun Tu ◽  
Anton Zilman ◽  
Siegfried M Musser
Keyword(s):  
Binding Sites ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Transport Receptors ◽  
Local Properties ◽  
Natively Unfolded ◽  
Mathematical Analyses ◽  
Super Resolution Microscopy ◽  
Source Of Information

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties.

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

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.

Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging

Science ◽  
2013 ◽  
Vol 341 (6146) ◽  
pp. 655-658 ◽  
Author(s):  
Anna Szymborska ◽  
Alex de Marco ◽  
Nathalie Daigle ◽  
Volker C. Cordes ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Super Resolution ◽  
Molecular Organization ◽  
Nuclear Pore ◽  
Protein Assemblies ◽  
Large Protein ◽  
Whole Cells ◽  
Molecular Machinery ◽  
Scaffold Structure ◽  
Super Resolution Microscopy

Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

Getting across the nuclear pore complex: new insights into nucleocytoplasmic transportThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 499-507 ◽  
Author(s):  
Daniel Stoffler ◽  
Kyrill Schwarz-Herion ◽  
Ueli Aebi ◽  
Birthe Fahrenkrog
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Special Issue ◽  
The Past ◽  
Transport Receptors ◽  
A Cell ◽  
New Evidence ◽  
Retention Signal ◽  
Selection Of

Small ions and molecules can traverse the nuclear pore complex (NPC) simply by diffusion, whereas larger proteins and RNAs require specific signals and factors that facilitate their passage through the NPC. Our understanding of the factors that participate and regulate nucleocytoplasmic transport has increased tremendously over the past years, whereas the actual translocation step through the NPC has remained largely unclear. Here, we present and discuss recent findings on the interaction between the NPC and transport receptors and provide new evidence that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargos.

scholarly journals Molecular determinants of large cargo transport into the nucleus

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Giulia Paci ◽  
Tiantian Zheng ◽  
Joana Caria ◽  
Anton Zilman ◽  
Edward A Lemke
Keyword(s):  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Cargo Transport ◽  
Transport Receptors ◽  
Molecular Determinants ◽  
Quantitative Understanding ◽  
Kinetics Of

Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex (NPC). Among the thousands of molecules that cross the NPC, even very large (>15 nm) cargoes such as pathogens, mRNAs and pre-ribosomes can pass the NPC intact. For these cargoes, there is little quantitative understanding of the requirements for their nuclear import, especially the role of multivalent binding to transport receptors via nuclear localisation sequences (NLSs) and the effect of size on import efficiency. Here, we assayed nuclear import kinetics of 30 large cargo models based on four capsid-like particles in the size range of 17–36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for nuclear transport can be recapitulated by a simple two-parameter biophysical model that correlates the import flux with the energetics of large cargo transport through the NPC. Together, our results reveal key molecular determinants of large cargo import in cells.

scholarly journals Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Kasper R Andersen ◽  
Evgeny Onischenko ◽  
Jeffrey H Tang ◽  
Pravin Kumar ◽  
James Z Chen ◽  
...  
Keyword(s):  
Nuclear Transport ◽  
Evolutionary Relationship ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Rotational Axis ◽  
Nuclear Pore Complexes ◽  
Facilitated Diffusion ◽  
Structural And Functional Properties ◽  
Transport Receptors ◽  
Pore Complexes

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery.

scholarly journals Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways

2012 ◽  
Vol 287 (15) ◽  
pp. 12277-12292 ◽  
Author(s):  
Poonam Malik ◽  
Alijan Tabarraei ◽  
Ralph H. Kehlenbach ◽  
Nadia Korfali ◽  
Ryota Iwasawa ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Host Cell ◽  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Nuclear Export ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Transport Pathways ◽  
Simplex Virus

The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin α/β-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways.

The dynamics of karyopherin-mediated nuclear transport

2001 ◽  
Vol 79 (5) ◽  
pp. 603-612 ◽  
Author(s):  
Marcello Marelli ◽  
David J Dilworth ◽  
Richard W Wozniak ◽  
John D Aitchison
Keyword(s):  
Biochemical Characterization ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Complex Interplay ◽  
Conventional View ◽  
Cytoplasmic Transport ◽  
Transport Receptors ◽  
Pore Complexes

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.Key words: Nucleocytoplasmic transport, nuclear pore complex, nucleoporin, karyopherin, Nup2p.

scholarly journals Ran-Binding Protein 3 Is a Cofactor for Crm1-Mediated Nuclear Protein Export

2001 ◽  
Vol 153 (7) ◽  
pp. 1391-1402 ◽  
Author(s):  
Mark E. Lindsay ◽  
James M. Holaska ◽  
Katie Welch ◽  
Bryce M. Paschal ◽  
Ian G. Macara
Keyword(s):  
Nuclear Protein ◽  
Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Yeast Protein ◽  
Permeabilized Cell ◽  
Ran Gtpase ◽  
Transport Receptors ◽  
Human Orthologue ◽  
Dependent Mechanism

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex posseses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1–RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase–activating protein.

Sign in / Sign up

Export Citation Format

Share Document