Cytoskeleton: Large-scale microtubule networks contract quite well

  1. Julio M Belmonte
  2. François Nédélec  Is a corresponding author
  1. European Molecular Biology Laboratory, Germany

The cytoskeleton of a cell plays many important roles, such as giving the cell its shape and organizing its contents. The filaments that make up the cytoskeleton assemble from protein monomers found in the cell’s cytoplasm. Two particularly important filament types for eukaryotic cells are actin filaments and microtubules, which both have crucial roles during various stages of cell division. For example, the mitotic spindle, which is essential for chromosome segregation, is made of microtubules. Motor proteins (for example myosin, kinesin and dynein) often work with these filaments to transport material across the cell and to form contracting structures such as muscles.

In the past decades, much effort has gone into characterizing the properties of microtubules, actin filaments and motor proteins, and their most important properties have probably been discovered already. However, we need a much better understanding of how all these components work together. Now, in eLife, Peter Foster, Sebastian Fürthauer, Michael Shelley and Daniel Needleman report the first quantitative study of an important process in this field of research – the contraction of microtubule networks (Foster et al., 2015).

Instead of relying on purified proteins to study how microtubules and motors organize (see, for example, Hentrich and Surrey, 2010), Foster et al. used extracts from frog eggs. These provide a more natural mixture of components and are commonly used to study the assembly of spindles (Sawin and Mitchison, 1991). They also performed the experiments in millimeter-wide channels, allowing them to finely control the overall geometry of the network. In all the experiments, drugs were used to promote the formation of stable microtubules and to prevent actin monomers assembling into filaments.

The microtubules initially formed in random configurations, and under the action of motor proteins assembled into star-shaped structures called asters, as previously reported (Hentrich and Surrey, 2010). The whole microtubule network then slowly contracted.

To clarify how these processes occurred, Foster and colleagues – who are based at Harvard University and New York University – used drugs to separately inhibit the activity of kinesin and dynein. This demonstrated that dynein accounts for 96% of the active stress in microtubule networks. Remarkably, carefully analyzing the contraction of the microtubule network also provided insights into actin biology. How is this possible?

While microtubule and the actin cytoskeleton are similar in many ways, there are important differences in the structures they form and the behaviors they display in vivo. Microtubules tend to form structures such as radial arrays because the filaments are few and tend to be straight due to their high rigidity. Moreover, since microtubules are often as long as the cell, the cell simply does not provide enough space to build the large microtubule networks that would be necessary for observing contraction. On the other hand, contraction is a common feature of actin networks, which can be made of many relatively short filaments that are 200 times more flexible than microtubules. These considerations reflect the fact that the behavior of a network is often largely a matter of scale: indeed, networks of filaments are usually analyzed in terms of filament length, the density of the filaments, and the overall size of the network (Lenz et al., 2012).

In the past, researchers have studied the contraction of actin networks at the micrometer scale. Now, Foster et al. were able to monitor the contraction of microtubule networks in millimeter-wide channels. Looking at the contractile behavior of filament networks in different regimes is especially valuable, because different contraction mechanisms are thought to operate at different scales. Actin network contractility is thought to require the bending of filaments, whereas microtubule contractility would rely on molecular motors holding tight to the ends of the microtubules (Figure 1). The ability to compare these two systems should improve our understanding of the general principles of contractility, and thus contribute to actin biology.

Two mechanisms for contraction: buckling and end clustering.

Top: When two anti-parallel actin filaments are bridged by a myosin motor (blue) and a crosslink (green), their relative movement forces one filament to buckle, resulting in the contraction of the network. Bottom: Microtubule contraction seems to depend on the affinity of dynein motors (red) for the ends of the filaments. For a recent review on the topic of contraction, see Clark et al., 2014.

Foster et al.’s approach may also teach us more about how mitotic spindles form. The molecular motor dynein, which induces the bulk contraction of large random networks, is also thought to help form the focused poles of the spindle. Specifically, contractions driven by dynein motors likely help the spindle to adopt the correct shape. Thus by carefully quantifying this contraction process, Foster et al. have likely given us some of the parameters needed to create accurate models of the mitotic spindle. For instance, the extract always contracted to the same final density, which is surprisingly similar to the density of the mitotic spindle. Future research could investigate the mechanism responsible for this density limit.

A remarkable aspect of the study is that Foster et al. could fit the bulk properties of the contraction with a simple active gel theory, using just four parameters. For example, the theory can explain how the microtubule density varies at the edge of the network and how the rate of contraction depends on the overall size of the network. This advance in our knowledge of cytoskeletal network contractility was only possible through a tight interplay between experiments and theory.

References

Article and author information

Author details

  1. Julio M Belmonte

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The author declares that no competing interests exist.
  2. François Nédélec

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    nedelec@embl.de
    Competing interests
    The author declares that no competing interests exist.

Publication history

  1. Version of Record published: February 12, 2016 (version 1)

Copyright

© 2016, Belmonte et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,549
    Page views
  • 278
    Downloads
  • 4
    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. Julio M Belmonte
  2. François Nédélec
(2016)
Cytoskeleton: Large-scale microtubule networks contract quite well
eLife 5:e14076.
https://doi.org/10.7554/eLife.14076
  1. Further reading

Further reading

    1. Structural Biology and Molecular Biophysics
    I Can Kazan, Prerna Sharma ... S Banu Ozkan
    Research Article

    We develop integrated co-evolution and dynamic coupling (ICDC) approach to identify, mutate, and assess distal sites to modulate function. We validate the approach first by analyzing the existing mutational fitness data of TEM-1 β-lactamase and show that allosteric positions co-evolved and dynamically coupled with the active site significantly modulate function. We further apply ICDC approach to identify positions and their mutations that can modulate binding affinity in a lectin, cyanovirin-N (CV-N), that selectively binds to dimannose, and predict binding energies of its variants through Adaptive BP-Dock. Computational and experimental analyses reveal that binding enhancing mutants identified by ICDC impact the dynamics of the binding pocket, and show that rigidification of the binding residues compensates for the entropic cost of binding. This work suggests a mechanism by which distal mutations modulate function through dynamic allostery and provides a blueprint to identify candidates for mutagenesis in order to optimize protein function.

    1. Structural Biology and Molecular Biophysics
    Jasenko Zivanov, Joaquín Otón ... Sjors HW Scheres
    Tools and Resources

    We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.