Nuclear pore function viewed with atomic force microscopy

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

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Quantification of Biomolecular Dynamics Inside Real and Synthetic Nuclear Pore Complexes Using Time-Resolved Atomic Force Microscopy

ACS Nano ◽

10.1021/acsnano.9b02424 ◽

2019 ◽

Vol 13 (7) ◽

pp. 7949-7956 ◽

Cited By ~ 2

Author(s):

George J. Stanley ◽

Bernice Akpinar ◽

Qi Shen ◽

Patrick D. Ellis Fisher ◽

C. Patrick Lusk ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Time Resolved ◽

Force Microscopy ◽

Atomic Force ◽

Biomolecular Dynamics ◽

Pore Complexes

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Atomic force microscopy reveals structural variability amongst nuclear pore complexes

Life Science Alliance ◽

10.26508/lsa.201800142 ◽

2018 ◽

Vol 1 (4) ◽

pp. e201800142 ◽

Cited By ~ 14

Author(s):

George J Stanley ◽

Ariberto Fassati ◽

Bart W Hoogenboom

Keyword(s):

Atomic Force Microscopy ◽

Intrinsically Disordered Proteins ◽

Structural Diversity ◽

Disordered Proteins ◽

Energetic Cost ◽

Nuclear Pore ◽

Central Channel ◽

Force Microscopy ◽

Intrinsically Disordered ◽

Atomic Force

The nuclear pore complex (NPC) is a proteinaceous assembly that regulates macromolecular transport into and out of the nucleus. Although the structure of its scaffold is being revealed in increasing detail, its transport functionality depends upon an assembly of intrinsically disordered proteins (called FG-Nups) anchored inside the pore's central channel, which have hitherto eluded structural characterization. Here, using high-resolution atomic force microscopy, we provide a structural and nanomechanical analysis of individual NPCs. Our data highlight the structural diversity and complexity at the nuclear envelope, showing the interplay between the lamina network, actin filaments, and the NPCs. It reveals the dynamic behaviour of NPC scaffolds and displays pores of varying sizes. Of functional importance, the NPC central channel shows large structural diversity, supporting the notion that FG-Nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Finally, different nuclear transport receptors are shown to interact in qualitatively different ways with the FG-Nups, with particularly strong binding of importin-β.

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