Influence of tubulin tyrosination on the localization of microtubule plus end tracking proteins and microtubule dynamics

Microtubules are the component the cytoskeleton involved in vital processes like migration, mitosis and intracellular trafficking. Alpha-tubulin is subjected to the tubulin tyrosination/detyrosination cycle, in which the C-terminal tyrosine residue is removed by a not yet characterized tubulin carboxy peptidase and subsequently re-added by tubulin tyrosine ligase (TTL). In this study, the influence of tubulin tyrosination on the localization of microtubule plus end tracking proteins (+TIPs) was analyzed. By using RNAi knock-down of +TIPs in mouse embryonic fibroblasts (MEFs), we found that depletion of EB1 leads to a TTL-dependent dislocalization of the CAP-Gly domain-containing protein CLIP-115 from the microtubule plus ends (MT +ends). CLIP-170 was removed from microtubule plus ends in both TTL wild type and TTL knock-out cells upon EB1 knock-down. In contrast, CLIP-115, a close homolog of CLIP-170 lacking the C-terminal zinc-binding domain, was dislocalized from microtubule plus ends only in wild type EB1-depleted cells. This CLIP-115 behavior could be rescued when TTL was reconstituted in TTL knock-out EB1-depleted MEFs. The observed difference in CLIP-115 MT +end binding is not caused by the lack of the C-terminal domain, since CLIP-170 mutant proteins lacking this domain localized to MT +ends similarly in both wild type and TTL-deficient MEFs. Analysis of microtubule binding of CLIP-115/170 hybrid proteins pointed to a possible role of coiled coil region in the distinct CLIP-115 MT binding. Moreover, TTL deficiency leads to changes in dynamics of microtubules. A decrease in microtubule shrinking speed as wells as an increase in the time microtubule spent pausing and a decrease in the time microtubule spent growing was observed. In addition TTL-deficiency resulted in increased levels of RhoA activity and a decrease of Rac1 and Cdc42 activity. Finally, the lack of TTL did not show any influence on the expression level of the RhoA effector mDia1.