Final Report Summary - MT-TIP INHIBITORS (Mechanisms and functions of microtubule plus end tracking proteins in mammalian cells: development of inhibitory strategies)
Microtubule targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and much less are known about how the effects of MTAs are modulated by microtubule-plus end associated proteins. In our study, we reconstituted microtubule dynamics in vitro to investigate the influence of EB proteins, the core components of the +TIP machinery, on the effects that MTAs exert on microtubule plus end growth. We found that EBs sensitise microtubules to the action of drugs both in cells and in vitro, by promoting catastrophes. This effect was irrespective of the nature of the binding site and the molecular mechanism of action of the MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age-dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways:
(a) destabilise MTAs, such as colchicine and vinblastine;
(b) accelerate aging in an EB-dependent manner, whereas they stabilise MTAs, such as paclitaxel and peloruside A;
(c) induce not only catastrophes but also rescuing and reversing the aging process.
We also developed inhibitory peptides to inhibit the interaction and functions of endogenous +TIP proteins and found them to efficiently track the plus ends of the microtubules and affect the cell division by arresting the cells at mitosis. Our attempt to generate cell permeable peptides was not successful, but it provided us new insights to develop efficient cell penetrating peptide inhibitors for plus end tracking. Overall, the study provided important insights into how the EB proteins modulate the effects of various drugs at physiological concentrations and the important role of these proteins in regulating microtubule functions.