15 research outputs found

    Taxanes convert regions of perturbed microtubule growth into rescue sites

    Get PDF
    Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anti -cancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization

    Inhibiting parasite proliferation using a rationally designed anti-tubulin agent

    Get PDF
    Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors. Natacha *#1, Ashwani *#1, Izra , Tianyang **3, Fiona **3, Alexander D. , Valentin , Mamata , Anthony J. , Carolyn A. , Naomi & Michel O.

    Comprehensive Analysis of Binding Sites in Tubulin

    No full text
    Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin\u2013protein and tubulin\u2013ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation

    Actin-microtubule coordination at growing microtubule ends

    No full text
    To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts

    Actin-microtubule coordination at growing microtubule ends

    No full text
    To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts

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

    No full text
    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 andmicrotubules,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 controlmultiple 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 onmicrotubule 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 aremicrotubule 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-dependentmanner,whereas stabilizingMTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process
    corecore