scholarly journals Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

2019 ◽  
Author(s):  
Thomas Schlichthaerle ◽  
Maximilian T. Strauss ◽  
Florian Schueder ◽  
Alexander Auer ◽  
Bianca Nijmeijer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Three Dimensions ◽  
Nuclear Pore ◽  
Technological Advancement ◽  
Molecular Architecture ◽  
Direct Visualization ◽  
Active Research ◽  
Molecular Contrast

The Nuclear Pore Complex (NPC) is one of the largest and most complex protein assemblies in the cell and – among other functions – serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

scholarly journals The Yeast Nuclear Pore Complex

2000 ◽  
Vol 148 (4) ◽  
pp. 635-652 ◽  
Author(s):  
Michael P. Rout ◽  
John D. Aitchison ◽  
Adisetyantari Suprapto ◽  
Kelly Hjertaas ◽  
Yingming Zhao ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Comprehensive Approach ◽  
Nuclear Pore ◽  
Molecular Architecture ◽  
Gating Mechanism ◽  
Molecular Components

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

scholarly journals Nuclear transport of single molecules

2005 ◽  
Vol 168 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Ulrich Kubitscheck ◽  
David Grünwald ◽  
Andreas Hoekstra ◽  
Daniel Rohleder ◽  
Thorsten Kues ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Far Field ◽  
Permeabilized Cells ◽  
Cytoplasmic Filaments ◽  
Transport Receptor

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex

1992 ◽  
Vol 50 (1) ◽  
pp. 490-491
Author(s):  
N. Panté ◽  
M. Jarnik ◽  
E. Heitlinger ◽  
U. Aebi
Keyword(s):  
Nuclear Pore Complex ◽  
Divalent Cations ◽  
Nuclear Lamina ◽  
Dynamic Structure ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Cytoplasmic Filaments ◽  
Radial Spokes ◽  
Nuclear Ring ◽  
Central Plug

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).

scholarly journals Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

2009 ◽  
Vol 185 (3) ◽  
pp. 475-491 ◽  
Author(s):  
Evgeny Onischenko ◽  
Leslie H. Stanton ◽  
Alexis S. Madrid ◽  
Thomas Kieselbach ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Partners ◽  
Interaction Partners ◽  
Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

Molecular architecture of the inner ring scaffold of the human nuclear pore complex

Science ◽  
2016 ◽  
Vol 352 (6283) ◽  
pp. 363-365 ◽  
Author(s):  
J. Kosinski ◽  
S. Mosalaganti ◽  
A. von Appen ◽  
R. Teimer ◽  
A. L. DiGuilio ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Molecular Architecture

scholarly journals Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.

1987 ◽  
Vol 104 (5) ◽  
pp. 1143-1156 ◽  
Author(s):  
C M Snow ◽  
A Senior ◽  
L Gerace
Keyword(s):  
Monoclonal Antibodies ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Integral Membrane Protein ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Punctate Pattern

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.

scholarly journals Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

1986 ◽  
Vol 102 (3) ◽  
pp. 859-862 ◽  
Author(s):  
M Schindler ◽  
L W Jiang
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Rabbit Serum ◽  
Nuclear Pore ◽  
Enhancement Effect ◽  
Flux Rate ◽  
Effective Transport ◽  
Liver Nuclei ◽  
Stimulation Of

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

Sign in / Sign up

Export Citation Format

Share Document