1. Structural Biology and Molecular Biophysics
  2. Cell Biology
Download icon

Investigating molecular crowding within nuclear pores using polarization-PALM

  1. Guo Fu
  2. Li-Chun Tu
  3. Anton Zilman
  4. Siegfried M Musser  Is a corresponding author
  1. The Texas A&M University Health Science Center, United States
  2. University of Toronto, Canada
Research Article
  • Cited 4
  • Views 2,320
  • Annotations
Cite this article as: eLife 2017;6:e28716 doi: 10.7554/eLife.28716

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.

Article and author information

Author details

  1. 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.
  2. 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.
  3. Anton Zilman

    Department of Physics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8523-6703
  4. 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.
    ORCID icon "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.

Reviewing Editor

  1. Karsten Weis, ETH Zurich, Switzerland

Publication history

  1. Received: May 18, 2017
  2. Accepted: September 25, 2017
  3. Accepted Manuscript published: September 26, 2017 (version 1)
  4. Version of Record published: November 17, 2017 (version 2)

Copyright

© 2017, Fu et al.

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.

Metrics

  • 2,320
    Page views
  • 456
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics
    Monica L Fernández-Quintero et al.
    Research Article Updated

    Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Agata Szuba et al.
    Research Article Updated

    Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.