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

Version 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

  • 3,009
    Page views
  • 426
    Downloads
  • 20
    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)

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. Eugene A Katrukha
  2. Daphne Jurriens
  3. Desiree M Salas Pastene
  4. Lukas C Kapitein
(2021)
Quantitative mapping of dense microtubule arrays in mammalian neurons
eLife 10:e67925.
https://doi.org/10.7554/eLife.67925

Share this article

https://doi.org/10.7554/eLife.67925

Further reading

    1. Cell Biology
    2. Plant Biology
    Maciek Adamowski, Ivana Matijević, Jiří Friml
    Research Article

    The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its closest homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study largely extend the current notions of GN function. We show that GN, but not GNL1, localizes to the cell periphery at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that the cell periphery is the major site of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations en route to the cell periphery. A study of GN-GNL1 chimeric ARF-GEFs indicates that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps toward the elucidation of the mechanism underlying unique cellular and development functions of GNOM.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Chenjie Xia, Huihui Xu ... Hongting Jin
    Research Article

    Glucocorticoid-induced osteonecrosis of the femoral head (GONFH) is a common refractory joint disease characterized by bone damage and the collapse of femoral head structure. However, the exact pathological mechanisms of GONFH remain unknown. Here, we observed abnormal osteogenesis and adipogenesis associated with decreased β-catenin in the necrotic femoral head of GONFH patients. In vivo and in vitro studies further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation of bone marrow mesenchymal cells (BMSCs) by inhibiting β-catenin signaling in glucocorticoid-induced GONFH rats. Col2+ lineage largely contributes to BMSCs and was found an osteogenic commitment in the femoral head through 9 mo of lineage trace. Specific deletion of β-catenin gene (Ctnnb1) in Col2+ cells shifted their commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice. Overall, we uncover that β-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of GONFH and identify an ideal genetic-modified mouse model of GONFH.