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.

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

  • 3,551
    views
  • 489
    downloads
  • 40
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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
    Satoshi Ninagawa, Masaki Matsuo ... Kazutoshi Mori
    Research Advance

    How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020; 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6α. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6α cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via the regulation of both glycoprotein folding and degradation.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Senem Ntourmas, Martin Sachs ... Dominic B Bernkopf
    Research Article

    Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.