Gold at microtubule tips

Essential components of our cells, microtubules resize permanently. One of the secrets of this dynamic instability resides in the stabilizing cap present at the tips of these structures. Led by scientists from the IGDR, a European collaboration revealed the architecture of this cap at the molecular level, as well as the conformational changes that take place in this key region of the microtubule. This work is published in Nature Cell Biology.

Gold nanoparticles (red) conjugated to the end-binding protein 1 (blue) at the tip of a microtubule (yellow).  Background: image of a section through a cryo-electron tomogram; gold nanoparticles are displayed in black and microtubules in grey. Credit: Guesdon, Bazile et al.

Microtubules are essential components of almost all eukaryotic cells. They are extremely dynamic biological polymers, some 25 nm in diameter and up to several tens of micrometers in length.

They assemble from a protein (the tubulin molecule) and are involved in several important cellular functions such as cytoplasm organization, cell motility and intracellular trafficking. Notably, they play a key role during cell division through the formation of the mitotic spindle that segregates equally chromosomes in daughter cells.

Dynamic instability and stabilizing cap

Amazingly, microtubules can alternate autonomously between growing and shrinking states. This behavior, called 'dynamic instability', relies on molecular mechanisms that are not fully understood.

Tubulin makes use of a small molecule (GTP) to provide energy to the microtubule. However, this energy is not used for microtubule assembly, but is stored as mechanical energy in their lattice, like a spring under tension: it follows that microtubules must be protected by a stabilizing cap at their extremity to prevent their fast depolymerization.

Until today, this cap has never been visualized at the molecular level.

Unveiling the microtubule stabilizing cap

To visualize the stabilizing cap present at the growing microtubule tips, scientists from the IGDR (team TIPs) conjugated gold nanoparticles to the EB1 protein (end-binding protein 1). Indeed, EB1 interacts preferentially with this region of the microtubule, but is too small to be visualized directly by electron microscopy.

To overcome this limitation, the scientists used gold nanoparticles attached to EB1, which can be effectively visualized by electron microscopy. They then froze rapidly growing microtubules in liquid ethane (-184°C).

Samples were then imaged by cryo-electron tomography. This imaging technique, still sparsely used in France, is developed by the team TIPs on the MRic platform from the Federative Institute Biosit. This method allows imaging complex macromolecular assemblages in their hydrated state and to perform three-dimensional reconstructions of these structures at the nanometer scale.

This work was undertaken during the thesis work of Dr. Audrey Guesdon and the post-doctoral position of Dr. Franck Bazile, both co-first authors of the manuscript.

 

Tubulin liganded to GTP (β-tubulin in blue) assembles as outwardly curved two-dimensional sheets at microtubule tips. These sheets gradually straighten and close into tubes. During this process, tubulin hydrolyses a GTP molecule into GDP (β-tubulin in red). This mechanism would involve a GDP-Pi intermediate (β-tubulin in yellow), which would participate to the architecture of the stabilizing cap. Following release of the inorganic phosphate (Pi) the GDP-tubulin lattice is in an unstable state, and must be protected by the GTP/GDP-Pi-tubulin cap at its growing extremity. Stochastic loss of this stabilizing structure would be at the origin of fast depolymerization events (catastrophes), and re-capping would allow re-polymerization (rescues).

Shedding light on anticancer drug mechanisms

Dynamic instability allows microtubules to reorganize rapidly upon cell needs. This mechanism is precisely tuned by an ensemble of proteins during the cell life and can also be modulated by pharmaceutical drugs. For instance, taxoids used in the treatment of some cancers bind directly to the stabilizing cap revealed during this study.

The team TIPs started a new collaboration with a pharmaceutical company (Esai Inc.), with the aim to localize at microtubule tips a small anti-mitotic molecule (Eribulin) used in the treatment of breast cancer and liposarcoma. This new project received already the support of the French 'Ligue Contre le Cancer' and of the University of Rennes 1.

Collaboration and reference

Collaboration

This work results from a collaboration between several European laboratories:

  • Dr. Denis Chrétien (Institute of Genetics and Development of Rennes), specialized in the analysis of macromolecular complexes by cryo-electron tomography.
  • Pr. Michel Steinmetz (Paul Scherrer Institut, Villigen, Switzerland), specialized in the analysis of proteins by X-ray crystallography.
  • Dr. Anna Akhmanova (Utrecht University, The Netherlands), specialized in the analysis of microtubule associated proteins by TIRF microscopy.
  • Pr. Robert Tampé (Goethe University, Frankfurt am Main, Germany), specialized in the chemistry of small ligands.
  • Dr. Rubén M. Buey (University of Salamanca, Spain), specialized in the biochemistry and molecular biology of tubulin.

Référence

EB1 interacts with outwardly curved and straight regions of the microtubule lattice
Audrey Guesdon, Franck Bazile, Rubén M. Buey, Renu Mohan, Solange Monier, Ruddi Rodríguez García, Morgane Angevin, Claire Heichette, Ralph Wieneke, Robert Tampé, Laurence Duchesne, Anna Akhmanova, Michel O. Steinmetz, Denis Chrétien
Nature Cell Biology doi:10.1038/ncb3142

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