End-binding proteins sensitize microtubules to the action of microtubule-targeting agents

Abstract

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all 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: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process. EB1 | EB3M icrotubule-targeting agents (MTAs) are a broad group of natural compounds and synthetic drugs that can promote assembly or disassembly of microtubules (MTs) at high concentrations and alter MT dynamics without significantly changing MT polymer mass at low concentrations (1, 2). These drugs are extensively used for the treatment of various cancers, and their effectiveness is mostly attributed to the ability to inhibit mitosis and disrupt tumor vasculature (1,3,4). Certain MTAs might also be candidates for treatment of other disorders, including neurodegenerative diseases (5).The wide use of MTAs and the continuous search for more efficacious and less toxic agents necessitates a detailed understanding of the mechanisms of their action. Biochemical and structural studies have revealed the major binding sites for MTAs on tubulin, including the vinca, colchicine, and taxane sites (1,3,6). At high concentrations, MTAs binding to the vinca and colchicine site destabilize MTs and decrease the polymer mass, and will here be referred to as MT-destabilizing drugs. Taxane sitebinding MTAs, such as paclitaxel, as well as some other drugs including peloruside A, promote MT stability and increase polymer mass, and will be referred to as MT-stabilizing drugs.In vitro studies with purified tubulin have indicated that at low concentrations some MTAs specifically interact with MT ends (1). Growing MT plus ends contain a stabilizing cap of GTP-bound tubulin; when this cap is lost, MTs switch to depolymerization, a transition termed catastrophe. Detailed analysis of MT growth parameters has shown that catastrophes depend on...

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

“…In particular, these mutants suppress catastrophe frequency by magnitudes similar to what is achieved by regulatory proteins or microtubule stabilizing drugs (Mohan et al, 2013; Wieczorek et al, 2015). Structural and mutagenesis studies have indicated that conformational changes in tubulin’s intermediate domain are coupled to GTP hydrolysis upon microtubule polymerization (Geyer et al, 2015; Nogales et al, 1998; Ravelli et al, 2004).…”

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

“…We speculate that the regulation of tubulin subunit on-off kinetics during microtubule growth and shortening constitutes a fundamental mechanism by which microtubuleassociated proteins and microtubule-targeting drugs regulate microtubule dynamics and nucleation (Mohan et al, 2013). Future studies to evaluate the effect of other microtubuleassociated proteins and drugs on microtubule assembly kinetics will be important for better understanding the mechanisms of the regulation of microtubule length in cells.…”

Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes

“…The effect of Eribulin-A488 on microtubule dynamics was tested using a total internal reflection fluorescence (TIRF) microscopybased microtubule dynamics reconstitution assay [20,21]. As shown in Figures 2D and 2E, 500 nM Eribulin strongly slows down the microtubule growth rate at plus ends without significantly affecting the catastrophe frequency.…”

“…Our previous work has shown that EBs sensitize microtubule plus ends to the action of microtubule-targeting agents by promoting catastrophes [21]. Similarly, we found that in the presence of mCherry-EB3, 50-100 nM Eribulin had a significant catastrophe-promoting effect at the plus end (Figures 2G and 2H), while five to ten times higher drug concentrations were needed to induce a profound effect on microtubule plus-end dynamics (reduction of growth rate) in the absence of mCherry-EB3 ( Figures 2D and 2E).…”