Cellular microtubule growth rates achieved in the test tube

Microtubules formed from purified tubulin in the test tube typically grow much more slowly than those in cells. In this study, we identified a minimal system, consisting only of tubulin and two regulatory proteins, sufficient to promote microtubule growth to cellular rates.

HFSP Cross-Disciplinary Fellow Marija Zanic and colleagues
authored on Fri, 31 May 2013

The microtubule network in cells undergoes rapid restructuring, which is dependent on the dynamic behavior of individual microtubules. Even in the test tube, microtubules assembled from purified tubulin switch between periods of growth and shrinkage, a phenomenon known as microtubule dynamic instability. However, the typical rates of growth achieved in vitro, as well as the rates of switching from growth to shrinkage (also known as ‘microtubule catastrophe’) are much lower than those observed in cells.

The observed difference between the behavior of microtubules in the test tube and those in cells is not surprising given the multitude of microtubule-associated proteins that regulate microtubule growth and catastrophe in cells. The effects of some of these proteins have been characterized using in vitro approaches. However, no individual protein has been shown to bring microtubule growth rates to physiological levels. Using a reconstitution assay with purified proteins and total-internal-reflection-fluorescence imaging, we have now identified a minimal system that leads to cellular microtubule growth rates. Our assay employs a combination of two microtubule-associated proteins, XMAP215 and EB1.

Figure: Kymographs depicting microtubule growth using purified tubulin in the presence and absence of XMAP215 and EB1. Dynamic microtubule extensions (green) are growing from stabilized microtubule seeds (red). Adapted from Zanic et al. NCB 2013.

XMAP215 is a well-known promoter of microtubule growth, which, on its own, increases the growth rates up to tenfold. EB1 is known for its ability to autonomously track growing microtubule ends, where it recruits many other microtubule regulators. When combined, these two proteins accelerate microtubule growth rates up to 20 µm/min, never previously observed in vitro. Remarkably, the effects of the two proteins are not purely additive; they act in synergy. While XMAP215 promotes growth by a factor of 10, and EB1 by 1.5, together they result in a 30-fold promotion of microtubule growth rates. We found that the synergy is not accomplished through a formation of a complex of the two proteins. Rather, our data and mathematical modeling predict that the synergy is a consequence of an allosteric interaction at the end of the growing microtubule.

Physiological microtubule growth rates in our reconstitution assay are accompanied by a high rate of catastrophe, rendering a strongly dynamic behavior typically observed only in cells. The discovery of synergy even in such a minimal system sheds light on the complex behaviors that arise in the network of microtubule regulating proteins. Integrating additional components of this network will be the next step toward understanding this important cellular system.

Reference

Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels. Zanic M, Widlund PO, Hyman AA, Howard J. Nat Cell Biol. 2013 doi: 10.1038/ncb2744.

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