Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

  1. Joseph M Cleary
  2. Tae Kim
  3. Annan SI Cook
  4. Lauren A McCormick
  5. William O Hancock  Is a corresponding author
  6. Luke M Rice  Is a corresponding author
  1. Pennsylvania State University, United States
  2. The University of Texas Southwestern Medical Center, United States

Abstract

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.

Data availability

Numerical data used to generate most of the figures have been provided as Source Data.

Article and author information

Author details

  1. Joseph M Cleary

    Department of Biomedical Engineering, Pennsylvania State University, University Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Tae Kim

    Departments of Biophysics and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Annan SI Cook

    Department of Biomedical Engineering, Pennsylvania State University, University Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Lauren A McCormick

    Departments of Biophysics and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9164-0932
  5. William O Hancock

    Department of Biomedical Engineering, Pennsylvania State University, University Park, United States
    For correspondence
    woh1@psu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5547-8755
  6. Luke M Rice

    Departments of Biophysics and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Luke.Rice@UTSouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6551-3307

Funding

National Science Foundation (MCB-1615938)

  • Tae Kim
  • Lauren A McCormick
  • Luke M Rice

National Institutes of Health (R01-GM135565)

  • Lauren A McCormick
  • Luke M Rice

National Institutes of Health (R35-GM139568)

  • Joseph M Cleary
  • William O Hancock

National Institutes of Health (T32-GM108563)

  • Joseph M Cleary

National Institutes of Health (T32-GM008297)

  • Tae Kim

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

Reviewing Editor

  1. Thomas Surrey, Centre for Genomic Regulation (CRG), Spain

Version history

  1. Preprint posted: November 25, 2021 (view preprint)
  2. Received: November 29, 2021
  3. Accepted: April 13, 2022
  4. Accepted Manuscript published: April 14, 2022 (version 1)
  5. Version of Record published: May 3, 2022 (version 2)

Copyright

© 2022, Cleary 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

  • 1,057
    Page views
  • 174
    Downloads
  • 5
    Citations

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

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

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

  1. Joseph M Cleary
  2. Tae Kim
  3. Annan SI Cook
  4. Lauren A McCormick
  5. William O Hancock
  6. Luke M Rice
(2022)
Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end
eLife 11:e75931.
https://doi.org/10.7554/eLife.75931

Further reading

    1. Cell Biology
    2. Microbiology and Infectious Disease
    Heledd Davies, Hugo Belda ... Moritz Treeck
    Tools and Resources

    Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

    1. Cell Biology
    Bo Wang, Zheyong Liang ... Peijun Liu
    Research Article

    The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.