1. Cell Biology
  2. Developmental Biology
Download icon

Quantitative mapping of dense microtubule arrays in mammalian neurons

Research Article
  • Cited 0
  • Views 1,033
  • Annotations
Cite this article as: eLife 2021;10:e67925 doi: 10.7554/eLife.67925

Abstract

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport.

Data availability

All quantitative data is available on Figshare:https://doi.org/10.6084/m9.figshare.c.5306546.v2Software is available on Zenodo:https://doi.org/10.5281/zenodo.4281064https://doi.org/10.5281/zenodo.4534715https://doi.org/10.5281/zenodo.4534721

Article and author information

Author details

  1. Eugene A Katrukha

    Department of Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  2. Daphne Jurriens

    Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  3. Desiree M Salas Pastene

    Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  4. Lukas C Kapitein

    Biology, Utrecht University, Utrecht, Netherlands
    For correspondence
    l.kapitein@uu.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9418-6739

Funding

H2020 European Research Council (819219)

  • Lukas C Kapitein

ZonMw (91217002)

  • Daphne Jurriens
  • Lukas C Kapitein

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

Ethics

Animal experimentation: Culturing of neurons has been approved by the ethical commitee (DEC) of Utrecht University and by the Centrale Commissie Dierproeven of the Dutch government (permit application AVD1080020173404). The ethical committee (DEC) is independent and must review any experimental use of animals in the Netherlands.

Reviewing Editor

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

Publication history

  1. Preprint posted: February 26, 2021 (view preprint)
  2. Received: February 26, 2021
  3. Accepted: July 24, 2021
  4. Accepted Manuscript published: July 27, 2021 (version 1)
  5. Version of Record published: September 3, 2021 (version 2)

Copyright

© 2021, Katrukha 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

  • 1,033
    Page views
  • 179
    Downloads
  • 0
    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
    Amit Kumar et al.
    Research Article Updated

    Hypoxic adaptation mediated by HIF transcription factors requires mitochondria, which have been implicated in regulating HIF1α stability in hypoxia by distinct models that involve consuming oxygen or alternatively converting oxygen into the second messenger peroxide. Here, we use a ratiometric, peroxide reporter, HyPer to evaluate the role of peroxide in regulating HIF1α stability. We show that antioxidant enzymes are neither homeostatically induced nor are peroxide levels increased in hypoxia. Additionally, forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1α protein stability. Moreover, decrease in lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD, failed to influence HIF1α protein stability. These data show that mitochondrial, cytosolic or lipid ROS were not necessary for HIF1α stability, and favor a model where mitochondria contribute to hypoxic adaptation as oxygen consumers.

    1. Cell Biology
    2. Developmental Biology
    Shaun Abrams, Jeremy F Reiter
    Research Article

    Craniofacial defects are among the most common phenotypes caused by ciliopathies, yet the developmental and molecular etiology of these defects is poorly understood. We investigated multiple mouse models of human ciliopathies (including Tctn2, Cc2d2a and Tmem231 mutants) and discovered that each displays hypotelorism, a narrowing of the midface. As early in development as the end of gastrulation, Tctn2 mutants displayed reduced activation of the Hedgehog (HH) pathway in the prechordal plate, the head organizer. This prechordal plate defect preceded a reduction of HH pathway activation and Shh expression in the adjacent neurectoderm. Concomitant with the reduction of HH pathway activity, Tctn2 mutants exhibited increased cell death in the neurectoderm and facial ectoderm, culminating in a collapse of the facial midline. Enhancing HH signaling by decreasing the gene dosage of a negative regulator of the pathway, Ptch1, decreased cell death and rescued the midface defect in both Tctn2 and Cc2d2a mutants. These results reveal that ciliary HH signaling mediates communication between the prechordal plate and the neurectoderm to provide cellular survival cues essential for development of the facial midline.