Abstract
Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.
Highlights
The dynamic nature of microtubules is critical for their function in cells
A delay in hydrolysis of unknown duration is thought to produce a 'GTP cap', a protective end structure formed of GTP-tubulins that is critical for microtubule stability [1,2,3,4,5]
How the biochemistry of GTP hydrolysis translates into conformational changes in the growing microtubule end, and thereby determines microtubule stability, is not understood [2, 7]
Summary
The dynamic nature of microtubules is critical for their function in cells. Microtubules polymerize by the addition of GTP-bound α/β-tubulin heterodimers to their ends. How the biochemistry of GTP hydrolysis translates into conformational changes in the growing microtubule end, and thereby determines microtubule stability, is not understood [2, 7]. EB3 decorated the entire lattice of GTPasedeficient E254A mutant microtubules (Fig. 2d-f), reminiscent of its binding to microtubules grown in the presence of the non-hydrolysable GTP analogue GTPγS 19.
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