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

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends

  1. Taylor A Reid
  2. Courtney Coombes
  3. Soumya Mukherjee
  4. Rebecca R Goldblum
  5. Kyle White
  6. Sneha Parmar
  7. Mark McClellan
  8. Marija Zanic
  9. Naomi Courtemanche
  10. Melissa K Gardner  Is a corresponding author
  1. University of Minnesota, United States
  2. Vanderbilt University, United States
Research Article
  • Cited 6
  • Views 1,967
  • Annotations
Cite this article as: eLife 2019;8:e48117 doi: 10.7554/eLife.48117

Abstract

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

Article and author information

Author details

  1. Taylor A Reid

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Courtney Coombes

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Soumya Mukherjee

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Rebecca R Goldblum

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Kyle White

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Sneha Parmar

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Mark McClellan

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Marija Zanic

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5127-5819
  9. Naomi Courtemanche

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Melissa K Gardner

    Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States
    For correspondence
    klei0091@umn.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5906-7363

Funding

National Institutes of Health (R01-GM103833)

  • Melissa K Gardner

National Institutes of Health (R35-GM126974)

  • Melissa K Gardner

National Science Foundation (1350741)

  • Melissa K Gardner

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

Reviewing Editor

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: May 1, 2019
  2. Accepted: August 23, 2019
  3. Accepted Manuscript published: September 3, 2019 (version 1)
  4. Version of Record published: September 12, 2019 (version 2)

Copyright

© 2019, Reid 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

  • 1,967
    Page views
  • 337
    Downloads
  • 6
    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. Cell Biology
    2. Neuroscience
    Friederike Elisabeth Kohrs et al.
    Tools and Resources

    Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous system-enriched Rabs yielded viable and fertile flies without obvious morphological defects. However, all 13 mutants differentially affected development when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. We identified a synaptic maintenance defect following continuous stimulation for six mutants, including an autophagy-independent role of rab26. The complete mutant collection generated in this study provides a basis for further comprehensive studies of Rab GTPases during development and function in vivo.

    1. Cell Biology
    Luca Minati et al.
    Tools and Resources Updated

    A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.