Atomic force microscopy reveals structural variability amongst nuclear pore complexes.

George J Stanley,Ariberto Fassati,Bart W Hoogenboom

doi:10.26508/lsa.201800142

George J Stanley, Ariberto Fassati + Show 1 more

Open Access

https://doi.org/10.26508/lsa.201800142

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Abstract

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.

Highlights

The nuclear pore complex (NPC) is a selective, nanoscale filter for macromolecules entering or exiting the nucleus
We show variability amongst the NPCs’ central channel, consistent with the prediction that FG-nuclear pore proteins (Nups) cohesiveness lies within a certain range such that the FG-Nups can alternate between different collective rearrangements at little energetic cost
atomic force microscopy (AFM) technology has developed new imaging modes, better control of the tip–sample interaction, and improved probe sharpness and consistency, facilitating AFM experiments on soft, biological samples in solution. We exploit these developments for imaging nuclear envelope (NE) at previously unattainable spatial resolution and reproducibility. This is exemplified on the cytoplasmic side of an NE (Fig 1A) that was mechanically isolated from a X. laevis oocyte

Summary

Introduction

The nuclear pore complex (NPC) is a selective, nanoscale filter for macromolecules entering or exiting the nucleus. Its central channel is occluded by natively disordered proteins, called FG-nucleoporins (or FG-Nups), which, in conjunction with soluble nuclear transport receptors (NTRs), form a selective barrier to transport This barrier is selective for molecules as small as ~4–5 nm in diameter (Mohr et al, 2009; Schmidt & Gorlich, 2016; Timney et al, 2016), and yet still permeable to others as large as ~39 nm (including human hepatitis B virus capsids) (Pante & Kann, 2002). Because the nucleocytoplasmic filtering mechanism resides in this FG-Nup assembly, our understanding of the NPC would benefit from a more thorough characterization of the FG-Nup morphology inside the central channel

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