Purified motor proteins organise microtubules in micrometre-sized droplets

Introduction

The control of the polymerisation/depolymerisation properties of microtubules is crucial for proper cytoskeleton functioning.

Dynamic microtubule properties are regulated in cells globally (e.g. responding to the cell cycle state) and also locally in response to local interactions.

It has become clear over the last 10 years that there are a multitude of proteins interacting selectively with microtubule ends to affect their dynamic properties. Several of these proteins interact with each other, thus forming a dynamic protein interaction network; this determines the fate of the microtubule and its interaction with other cellular structures. How this protein network functions as a system is not understood.

Using an in vitro reconstitution approach combined with total internal reflection (TIRF) microscopy, we have recently elucidated the molecular mechanism by which the key member (EB1) of this local protein interaction network localises specifically at microtubule ends, and how it recruits some of the other proteins of this network (Bieling et al., Nature, 2007; Bieling et al., J. Cell Biol., 2008)

We have shown that a specific conformation of the tubulin subunits exists over an extended region at growing microtubule ends to which EB1 binds. Interestingly this is a conformation related to the GTP cap (Maurer et al., PNAS, 2011), and EB1 binds close to the hydrolysable GTP (Maurer, Fourniol et al., Cell, 2012). Using microfluidics, we demonstrated that longer caps make microtubules more stable (Duellberg et al., eLife, 2016) and cap size fluctuations can be understood quantitatively based on the reaction network of cap formation (Rickman, PNAS, 2017). Presently, we investigate how these fluctuations and the nanoscale structure of microtubule ends relate to the fundamental property of dynamic microtubule instability.

Surrey columns

Cryo-electron microscopy reconstruction of the microtubule binding domain of EB1 on a microtubule.
Cryo-electron microscopy reconstruction of the microtubule binding domain of EB1 on a microtubule (Maurer, Fourniol et al., Cell 2012). 
 TIRF microscopy image sequence
 TIRF microscopy image sequence (Duellberg et al., eLife, 2016).
 Time plots of the microtubule end position
Time plots of the microtubule end position (red) and the corresponding EB1-GFP intensity (green) in a tubulin washout experiment (Duellberg et al., eLife, 2016). Scale bar is 3 µm.
Scatter plot of delay times between washout and catastrophe.
Scatter plot of delay times between washout and catastrophe versus EB1-GFP intensities at the time of tubulin washout, showing a correlation between instantaneous microtubule stability and protective cap size.