Investigating molecular crowding within nuclear pores using polarization-PALM

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Abstract

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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Guo Fu

Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M University Health Science Center, College Station, United States

Competing interests
The authors declare that no competing interests exist.
Li-Chun Tu

Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M University Health Science Center, College Station, United States

Competing interests
The authors declare that no competing interests exist.
Anton Zilman

Department of Physics, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8523-6703
Siegfried M Musser

Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M University Health Science Center, College Station, United States

For correspondence
smusser@tamhsc.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7793-2557

Funding

National Institutes of Health (GM084062)

Siegfried M Musser

Welch Foundation (BE-1541)

Siegfried M Musser

Canadian National Science and Engineering Research Council (RGPIN-2016-06591)

Anton Zilman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.