Dynamic life of a microtubule: From birth, growth and stabilization to damage and destruction

Abstract

Microtubules are one of the major types of cytoskeletal filaments in cells. They are very dynamic polymers composed of αβ-tubulin dimers arranged longitudinally in head-to-tail fashion as well as laterally to assemble 13-protofilament hollow cylindrical tubes. The incorporation of GTP-bound αβ-tubulin dimers generates a fast growing plus end exposing β-tubulin and a slow growing minus end exposing α-tubulin. In cells, microtubules are assembled de novo from a template, called γ-TuRC, which interacts with α-tubulin. Microtubules can either remain capped by γ-TuRC and anchored to the microtubule-organizing centers (MTOCs) or be released if they are cut by microtubule severing enzymes like katanin. The release of microtubules from MTOC generates free minus ends, which are then stabilized by minus-end binding proteins called CAMSAPs. However, the plus ends remain very dynamic and undergo transitions from growth to shrinkage, termed “catastrophes”, and the opposite transitions termed “rescues”. Numerous microtubule regulatory proteins act at the plus ends, minus ends and the microtubule shafts connecting the two ends to control the organization and density of cellular microtubule networks. In this thesis, we focused on each of these aspects and explored the dynamic life of microtubules by reconstituting these processes in vitro using purified proteins. We first focused on the birth and growth of microtubules. We reconstituted microtubule nucleation using purified γ-TuRC and microtubule regulatory proteins and showed that CDK5RAP2, CLASP2 and chTOG promoted microtubule nucleation from γ-TuRC. We discovered that CAMSAPs can bind to γ-TuRC-capped microtubule minus ends and displace γ-TuRC from these ends, generating free and stable microtubule minus ends. Furthermore, we found out that CDK5RAP2, but not CLASP2 or chTOG, can inhibit CAMSAP binding and microtubule release. We propose that the destiny of a microtubule depends on the type of protein complex that activates its nucleation. We then described a mechanism for stabilization of microtubule lattice by TRIM46, a neuronal protein, which can bundle parallel microtubules and promote microtubule rescues within these bundles. We also revealed that Ankyrin-G, a scaffold protein, can recruit TRIM46-stabilized microtubule bundles to the axonal membrane to drive the assembly of the axon initial segment in neurons. We also uncovered a new role of CLASP2 as a microtubule repair factor participating in microtubule maintenance. We demonstrated that CLASP2, an anti-catastrophe factor, can promote complete repair of damaged microtubule lattices by inhibiting microtubule depolymerization and promoting tube closure at the damage sites, causing lattice renewal. Finally, we described a three-protein module involving katanin, CAMSAPs, and WDR47 that can regulate microtubule polymer mass and minus-end stability. We showed that katanin can cut and amplify CAMSAP2/3-stabilized microtubule minus ends. WDR47 can inhibit the binding of katanin to CAMSAP2/3-stabilized minus ends and protect them from severing. The presence of WDR47 shifts the balance from microtubule amplification to minus-end growth regulation. To conclude, we obtained mechanistic insights into the regulation of microtubule nucleation, minus-end dynamics, lattice stabilization and maintenance, microtubule number and the interplay between microtubule regulatory proteins. These insights will help to understand how microtubule arrays are organized in cells.