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Mechanisms and functions of microtubule plus end tracking proteins in mammalian cells: development of inhibitory strategies

Final Report Summary - MT-TIP INHIBITORS (Mechanisms and functions of microtubule plus end tracking proteins in mammalian cells: development of inhibitory strategies)

Microtubules play vital role in various cellular functions such as cell division, intracellular transport, cell migration etc. Plus-end tracking proteins (+TIPs) are a specialised group of microtubule-associated factors that bind to the growing ends of microtubules and regulate various functions of microtubules. End binding (EB) proteins form the core of the +TIP interaction network, that links these proteins to the growing plus ends of the microtubule. Most of the +TIP proteins have a short conserved SXIP motif, that is involved in EB-dependent microtubule plus end localisation. The goal of the project was to use various inhibitory strategies (namely, anti-microtubule drugs or inhibitory peptides) targeting EB-microtubule or EB-SXIP interaction to gain insight into the +TIP-microtubule interaction, +TIP function and antiproliferactive actions of the microtubule targeted drugs. The project also aimed to develop cell-permeable peptides as markers for plus ends and / or potential inhibitors of plus end tracking.

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.