The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

Abstract

Microtubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes 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 regulatory hub that modulates microtubule cytoskeleton function in cells.