A coarse-grained computational model of the nuclear pore complex predicts Phe-Gly nucleoporin dynamics

The phenylalanine-glycine–repeat nucleoporins (FG-Nups), which occupy the lumen of the nuclear pore complex (NPC), are critical for transport between the nucleus and cytosol. Although NPCs differ in composition across species, they are largely conserved in organization and function. Transport through the pore is on the millisecond timescale. Here, to explore the dynamics of nucleoporins on this timescale, we use coarse-grained computational simulations. These simulations generate predictions that can be experimentally tested to distinguish between proposed mechanisms of transport. Our model reflects the conserved structure of the NPC, in which FG-Nup filaments extend into the lumen and anchor along the interior of the channel. The lengths of the filaments in our model are based on the known characteristics of yeast FG-Nups. The FG-repeat sites also bind to each other, and we vary this association over several orders of magnitude and run 100-ms simulations for each value. The autocorrelation functions of the orientation of the simulated FG-Nups are compared with in vivo anisotropy data. We observe that FG-Nups reptate back and forth through the NPC at timescales commensurate with experimental measurements of the speed of cargo transport through the NPC. Our results are consistent with models of transport where FG-Nup filaments are free to move across the central channel of the NPC, possibly informing how cargo might transverse the NPC.

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Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex

The Journal of Cell Biology ◽

10.1083/jcb.201105042 ◽

2011 ◽

Vol 195 (2) ◽

pp. 183-192 ◽

Cited By ~ 6

Author(s):

Philipp Stelter ◽

Ruth Kunze ◽

Jessica Fischer ◽

Ed Hurt

Keyword(s):

Nuclear Pore Complex ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Binding Motif ◽

Dynein Light Chain ◽

Transport Channel ◽

Functional Features ◽

And Function ◽

Insight Into

Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain–interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

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Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

The Journal of Cell Biology ◽

10.1083/jcb.129.5.1205 ◽

1995 ◽

Vol 129 (5) ◽

pp. 1205-1216 ◽

Cited By ~ 38

Author(s):

H Mehlin ◽

B Daneholt ◽

U Skoglund

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Pore ◽

Initial Contact ◽

Chironomus Tentans ◽

Central Channel ◽

Balbiani Ring ◽

Salivary Gland Cells ◽

Larval Salivary Gland ◽

Nuclear Ring ◽

Precise Area

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

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Allosteric Regulation in Gating the Central Channel of the Nuclear Pore Complex

Cell ◽

10.1016/j.cell.2015.05.013 ◽

2015 ◽

Vol 161 (6) ◽

pp. 1361-1373 ◽

Cited By ~ 30

Author(s):

Junseock Koh ◽

Günter Blobel

Keyword(s):

Nuclear Pore Complex ◽

Allosteric Regulation ◽

Nuclear Pore ◽

Central Channel

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Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200608141 ◽

2006 ◽

Vol 175 (4) ◽

pp. 579-593 ◽

Cited By ~ 100

Author(s):

Benjamin L. Timney ◽

Jaclyn Tetenbaum-Novatt ◽

Diana S. Agate ◽

Rosemary Williams ◽

Wenzhu Zhang ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Pore Complex ◽

Nuclear Import ◽

Yeast Cells ◽

Limiting Factor ◽

Nuclear Pore ◽

Nuclear Localization Signals ◽

The Poor ◽

Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

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An agent-based model for mRNA export through the nuclear pore complex

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1065 ◽

2014 ◽

Vol 25 (22) ◽

pp. 3643-3653 ◽

Cited By ~ 16

Author(s):

Mohammad Azimi ◽

Evgeny Bulat ◽

Karsten Weis ◽

Mohammad R. K. Mofrad

Keyword(s):

Nuclear Pore Complex ◽

Mrna Export ◽

Nuclear Pore ◽

Central Channel ◽

Agent Based Model ◽

Agent Based ◽

Transport Dynamics ◽

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.

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Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex

Structure ◽

10.1016/j.str.2013.02.005 ◽

2013 ◽

Vol 21 (4) ◽

pp. 560-571 ◽

Cited By ~ 39

Author(s):

Parthasarathy Sampathkumar ◽

Seung Joong Kim ◽

Paula Upla ◽

William J. Rice ◽

Jeremy Phillips ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Pore ◽

Structure Dynamics ◽

And Function

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Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein.

The Journal of Cell Biology ◽

10.1083/jcb.116.1.15 ◽

1992 ◽

Vol 116 (1) ◽

pp. 15-30 ◽

Cited By ~ 61

Author(s):

U F Greber ◽

L Gerace

Keyword(s):

Nuclear Pore Complex ◽

Protein Import ◽

Nuclear Protein ◽

Complex Structure ◽

Passive Diffusion ◽

Nuclear Pore ◽

Transport Inhibition ◽

Cell Progression ◽

Pore Complexes

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.

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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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NUP50 is necessary for the survival of primordial germ cells in mouse embryos

Reproduction ◽

10.1530/rep-14-0649 ◽

2016 ◽

Vol 151 (1) ◽

pp. 51-58 ◽

Cited By ~ 5

Author(s):

Eunsook Park ◽

Bobae Lee ◽

Bruce E Clurman ◽

Keesook Lee

Keyword(s):

Embryonic Development ◽

Germ Cells ◽

Nuclear Pore Complex ◽

Essential Role ◽

Primordial Germ Cells ◽

Mouse Embryos ◽

Nuclear Pore ◽

Progressive Loss ◽

And Function ◽

Deficient Mice

Nucleoporin 50 kDa (NUP50), a component of the nuclear pore complex, is highly expressed in male germ cells, but its role in germ cells is largely unknown. In this study, we analyzed the expression and function of NUP50 during the embryonic development of germ cells using NUP50-deficient mice. NUP50 was expressed in germ cells of both sexes at embryonic day 15.5 (E15.5), E13.5, and E12.5. In addition, NUP50 expression was also detected in primordial germ cells (PGCs) migrating into the genital ridges at E9.5. The gonads of Nup50−/− embryos of both sexes contained few PGCs at both E11.5 and E12.5 and no developing germ cells at E15.5. The migratory PGCs in Nup50−/− embryos at E9.5 showed increased apoptosis but a normal rate of proliferation, resulting in the progressive loss of germ cells at later stages. Taken together, these results suggest that NUP50 plays an essential role in the survival of PGCs during embryonic development.

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