Faculty Opinions recommendation of Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.

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.

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Faculty Opinions recommendation of Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718029894.793483627 ◽

2013 ◽

Author(s):

Thomas Schwartz

Keyword(s):

Super Resolution ◽

Nuclear Pore ◽

Scaffold Structure ◽

Super Resolution Microscopy

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Super-Resolution Microscopy Gives New Insights into Nuclear Pore Complex Organization

BioTechniques ◽

10.2144/000114221 ◽

2014 ◽

Vol 57 (4) ◽

Keyword(s):

Nuclear Pore Complex ◽

Super Resolution ◽

Nuclear Pore ◽

Complex Organization ◽

Super Resolution Microscopy

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Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy

Scientific Reports ◽

10.1038/s41598-017-15433-2 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 12

Author(s):

Julien Sellés ◽

May Penrad-Mobayed ◽

Cyndélia Guillaume ◽

Alica Fuger ◽

Loïc Auvray ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Developmental Process ◽

Super Resolution ◽

Nuclear Pore ◽

Super Resolution Microscopy

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3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e20-11-0728 ◽

2021 ◽

pp. mbc.E20-11-0728

Author(s):

Vilma Jimenez Sabinina ◽

M. Julius Hossain ◽

Jean-Karim Hériché ◽

Philipp Hoess ◽

Bianca Nijmeijer ◽

...

Keyword(s):

3D Structure ◽

Super Resolution ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Molecular Architecture ◽

Nuclear Ring ◽

Molecular Specificity ◽

Pore Complexes ◽

Super Resolution Microscopy ◽

Exciting Possibility

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.

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Investigating molecular crowding within nuclear pores using polarization-PALM

eLife ◽

10.7554/elife.28716 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 5

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.

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Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication

10.1101/664409 ◽

2019 ◽

Author(s):

Nidhi Vishnoi ◽

Karthigeyan Dhanasekeran ◽

Madeleine Chalfant ◽

Ivan Surovstev ◽

Mustafa K. Khokha ◽

...

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

Congenital Heart ◽

Super Resolution ◽

State Level ◽

Fluorescent Labeling ◽

Nuclear Pore ◽

Centriole Duplication ◽

Pericentriolar Material ◽

Super Resolution Microscopy

AbstractNUP188 encodes a scaffold component of the nuclear pore complex (NPC) and has been implicated as a congenital heart disease gene through an ill-defined function at centrioles. Here, we explore the mechanisms that physically and functionally segregate Nup188 between the pericentriolar material (PCM) and NPCs throughout the cell cycle. Pulse-chase fluorescent labeling approaches indicate that Nup188 populates centrosomes with newly synthesized protein that does not exchange with NPCs even after mitotic NPC breakdown. In addition, the steady-state level of Nup188 at centrosomes is controlled by the sensitivity of the PCM pool, but not the NPC pool, to proteasomal degradation. Proximity-labeling and super-resolution microscopy supports that Nup188 interacts with components of PCM including Cep192 and the centriolar satellite component, PCM1. Consistent with this, Nup188 plays a role in centriole duplication at or upstream of Sas6 loading. Together, our data establish Nup188 as a functional component of PCM and potentially provides insight into the pathogenesis of congenital heart disease.

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Single mRNP analysis by super-resolution microscopy and fluorescence correlation spectroscopy reveals that small mRNP granules represent mRNA singletons

10.1101/558098 ◽

2019 ◽

Author(s):

Àngels Mateu-Regué ◽

Jan Christiansen ◽

Frederik Otzen Bagger ◽

Christian Hellriegel ◽

Finn Cilius Nielsen

Keyword(s):

Translational Control ◽

Fluorescence Correlation Spectroscopy ◽

Rna Binding ◽

Rna Binding Proteins ◽

Super Resolution ◽

Correlation Spectroscopy ◽

Live Cells ◽

Nuclear Pore ◽

Fluorescence Correlation ◽

Super Resolution Microscopy

SummarySmall cytoplasmic mRNP granules are implicated in mRNA transport, translational control and decay. Employing Super-resolution Microscopy and Fluorescence Correlation Spectroscopy, we analyzed the molecular composition and dynamics of single cytoplasmic YBX1_IMP1 mRNP granules in live cells. Granules appeared elongated and branched with patches of IMP1 and YBX1 distributed along mRNA, reflecting the attachment of the two RNA-binding proteins in cis. Particles form at the nuclear pore and are spatially segregated from translating ribosomes, so the mRNP is a repository for mRNAs awaiting translation. In agreement with the average number of mRNA-binding sites derived from CLIP analyses, individual mRNPs contain 5 to 15 molecules of YBX1 and IMP1 and a single poly(A) tail identified by PABPC1. Taken together, we conclude that small cytoplasmic mRNP granules are mRNA singletons, thus depicting the cellular transcriptome. Consequently, expression of functionally related mRNAs in RNA regulons is unlikely to result from coordinated assembly.

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3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

10.1101/2020.11.27.386599 ◽

2020 ◽

Author(s):

Vilma Jimenez Sabinina ◽

M. Julius Hossain ◽

Jean-Karim Hériché ◽

Philipp Hoess ◽

Bianca Nijmeijer ◽

...

Keyword(s):

3D Structure ◽

Super Resolution ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Molecular Architecture ◽

Nuclear Ring ◽

Molecular Specificity ◽

Pore Complexes ◽

Super Resolution Microscopy ◽

Exciting Possibility

AbstractNuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.

Download Full-text