Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant

  1. Alessandro Dema
  2. Rabab A Charafeddine
  3. Shima Rahgozar
  4. Jeffrey van Haren
  5. Torsten Wittmann  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Erasmus MC, Netherlands

Abstract

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here, we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step, thereby enabling this approach in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.

Data availability

Raw data have been deposited to Dryad: doi:10.7272/Q6CF9NC5

The following data sets were generated

Article and author information

Author details

  1. Alessandro Dema

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0976-9396
  2. Rabab A Charafeddine

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Shima Rahgozar

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jeffrey van Haren

    Department of Cell Biology, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3160-3547
  5. Torsten Wittmann

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    For correspondence
    Torsten.Wittmann@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9134-691X

Funding

National Cancer Institute (R21 CA224194)

  • Torsten Wittmann

National Institute of Neurological Disorders and Stroke (R01 NS107480)

  • Torsten Wittmann

National Institutes of Health (S10 RR026758)

  • Torsten Wittmann

National Institutes of Health (S10 OD028611)

  • Torsten Wittmann

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

Reviewing Editor

  1. Kassandra M Ori-McKenney, University of California, United States

Version history

  1. Preprint posted: September 23, 2022 (view preprint)
  2. Received: October 12, 2022
  3. Accepted: January 27, 2023
  4. Accepted Manuscript published: January 30, 2023 (version 1)
  5. Version of Record published: February 10, 2023 (version 2)

Copyright

© 2023, Dema 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.

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  1. Alessandro Dema
  2. Rabab A Charafeddine
  3. Shima Rahgozar
  4. Jeffrey van Haren
  5. Torsten Wittmann
(2023)
Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant
eLife 12:e84143.
https://doi.org/10.7554/eLife.84143

Share this article

https://doi.org/10.7554/eLife.84143

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