Abstract

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Akanksha Thawani

    Department of Chemical and Biological Engineering, Princeton University, Princeton, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Howard A Stone

    Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, United States
    For correspondence
    hastone@princeton.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9670-0639
  3. Josh W Shaevitz

    Department of Physics, Princeton University, Princeton, United States
    For correspondence
    shaevitz@princeton.edu
    Competing interests
    The authors declare that no competing interests exist.
  4. Sabine Petry

    Department of Molecular Biology, Princeton University, Princeton, United States
    For correspondence
    spetry@Princeton.EDU
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8537-9763

Funding

American Heart Association (17PRE33660328)

  • Akanksha Thawani

National Institute of General Medical Sciences (1DP2GM123493-01)

  • Sabine Petry

Pew Charitable Trusts (00027340)

  • Sabine Petry

David and Lucile Packard Foundation (2014-40376)

  • Sabine Petry

National Science Foundation (PHY-1734030)

  • Josh W Shaevitz

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

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocol # 1941-16 of Princeton University.

Copyright

© 2019, Thawani 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

  • 4,688
    views
  • 741
    downloads
  • 55
    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. Akanksha Thawani
  2. Howard A Stone
  3. Josh W Shaevitz
  4. Sabine Petry
(2019)
Spatiotemporal organization of branched microtubule networks
eLife 8:e43890.
https://doi.org/10.7554/eLife.43890

Share this article

https://doi.org/10.7554/eLife.43890

Further reading

    1. Cell Biology
    2. Genetics and Genomics
    Jisun So, Olivia Strobel ... Hyun Cheol Roh
    Tools and Resources

    Single-nucleus RNA sequencing (snRNA-seq), an alternative to single-cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying mouse adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

    1. Cell Biology
    Inês Sequeira
    Insight

    A combination of intermittent fasting and administering Wnt3a proteins to a bone injury can rejuvenate bone repair in aged mice.