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Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

Davide Gambarotto, Fabian Zwettler, Marketa Cernohorska, Denis Fortun, Susanne Borgers, Jorn Heine, Jan-Gero Schloetel, Matthias Reuss, Michael Unser, Edward Boyden, Markus Sauer, Virginie Hamel, Paul Guichard

Preprint posted on April 25, 2018 https://www.biorxiv.org/content/early/2018/04/25/308270

Go beyond the limits! Gambarotto et al. report a novel method of near-native expansion microscopy (UltraExM), which goes beyond the super-resolution limits and reveals ultrastructural details of centrioles.

Selected by Satish Bodakuntla

Categories: cell biology

Context: Availability of technical tools has always been limiting our ability to advance in science, especially biology. Understanding a cellular component/protein is often restricted by how well we know its physiological location. Despite several landmark technical advances (electron microscopy, STED, SIM, STORM, PALM and ExM) that significantly improved our knowledge of cellular systems, we are yet to fully comprehend the ultra-structural details of a cell. In this preprint, the authors developed ultrastructure ExM (UltraExM), a novel expansion microscopy method that preserves and allows visualizing the molecular architecture of multiprotein complexes at an ultrastructural level.

Key findings: The authors systematically compared the performance of established expansion microscopy techniques (physically expanding the sample) on centrioles from Chlamydomonas. They showed how these techniques are unable to uncover the fine details of centrioles without compromising their macromolecular architecture. To overcome this limitation, they have developed a novel method of expansion microscopy that sheds light on the architectural details of centrioles and on the precise localization of tubulin polyglutamylation, posttranslational modification of microtubules.

  1. UltraExM combined with super resolution microscopy allowed the authors to visualize the 9-fold symmetry of the microtubules and more importantly their centriolar chirality; such analyses until now have largely relied on electron microscopy.
  2. Using this technique, the authors for the first time unveiled the precise localization of tubulin polyglutamylation on centrioles. Polyglutamylation was shown to be around the centriole microtubules, which possibly explains how this modification is implicated in the stability and maturation of centrioles. Another exciting discovery in the preprint was to show the sub-microtubule triplet localization of tubulin polyglutamylation.

Why this preprint is interesting: This preprint describes a novel method that allows visualizing the ultrastructural details of the organelles. Their systematic evaluation of various existing methods on visualizing the protein complexes is remarkable. Further, their methodical optimization of Magnified Analysis of Proteome (MAP) protocol to achieve intact centriolar expansion with precise dimensions is impressive. I believe that this method will bring great benefit to biologists who are interested in understanding the fine details of the cellular structures. 

Questions the work raises:

The authors used Chlamydomonas centrioles with known measurements to compare the performance of existing methods and to standardize the conditions for UltraExM. Can this method be applied only on protein complexes/organelles with known dimensions?

Using this novel method, it will be interesting to see how centriolar polyglutamylation is altered in disease conditions and it effects the centriole-protein interactions.

 

Tags: centriolar chirality, centrioles, super-resolution microscopy, tubulin polyglutamylation, ultraexm

Posted on: 10th July 2018

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  • Author's response

    Paul Guichard and Virginie Hamel shared

    The authors used Chlamydomonas centrioles with known measurements to compare the performance of existing methods and to standardize the conditions for UltraExM. Can this method be applied only on protein complexes/organelles with known dimensions?

    Yes. We have extended our study and now showed that it works on other macromolecular assemblies such as the cilia and viruses.

     

    Using this novel method, it will be interesting to see how centriolar polyglutamylation is altered in disease conditions and it effects the centriole-protein interactions.

    Yes, using UltraExM it would be possible indeed to follow centriolar polyglutamylation and/or other centriolar proteins in different conditions, including disease, mutants or RNAi.

    1 comment

    2 months

    Satish Bodakuntla

    Thank you Paul and Virginie for sharing your thoughts on the questions raised in the highlight.




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