Tipping microtubule polymerization: Insights from in vitro reconstitution

Abstract

Microtubules are biopolymers composed of αβ-tubulin dimers that associate in a head-to-tail fashion into protofilaments, which interact laterally to form a tube. Tubulin dimers are incorporated at the growing microtubule ends in GTP-bound form. Within a polymerized microtubule lattice, GTP is hydrolysed to GDP, but this process occurs with a time delay. This leads to the formation of a transient GTP cap that stabilizes the growing microtubule, whereas its loss results in a switch to shrinkage, termed catastrophe. Control of the transitions from microtubule growth to shrinkage is critical for fundamental processes like cell division, morphogenesis and migration. Over the years, through a combination of structural, biophysical and cellular approaches, significant progress has been achieved in understanding of the regulation of microtubule dynamics by cellular factors. In this thesis, we shed light on the activities of multiple mammalian microtubule plus end binding proteins in the regulation of this switching behaviour using in vitro reconstitutions with purified components. We find that the mammalian microtubule plus end complexes include two modules - a growth-stabilizing module with EB, CLASP and CLIP-170, and a polymerization-promoting module consisting of chTOG, SLAIN2 and EB that stimulates fast growth but reduces microtubule stability. In this thesis, we showed that different protein assemblies regulate distinct aspects of microtubule dynamics by virtue of their specialized domains and interactions. This study highlights the complexity of the molecular mechanisms utilised by microtubule associated proteins, which determine the architecture of microtubule arrays by regulating microtubule dynamics at the plus end.