A biophysical approach to elucidating the molecular mechanisms of mitotic inhibitor targets

许多化疗药物以微管为靶点，以抑制有丝分裂和阻止细胞增殖 癌细胞。一些有丝分裂抑制剂改变微管组装动力学，以延缓或阻止微管 另一些则阻止微管附着在相关的有丝分裂机制上。然而， 微管生长和微管附着到动点的基本过程仍然很差 明白了。这项提议的目的是阐明有丝分裂抑制物的分子靶标是如何在 以及抗癌疗法如何改变其作用机制。 在这项研究中使用的中心方法是用纯化的 重组蛋白。这种自下而上的方法具有允许直接和完全控制的优点 在实验条件下，以及能够使用强大的 分析技术。在这项建议中，一种名为干涉散射显微镜的新显微技术 (ISCAT)被改进和应用，以回答以微管为中心的机制问题。ISCAT 支持以高达每秒50,000帧的帧速率直接显示未标记的微管，并且可以 测量用30纳米金纳米颗粒标记的蛋白质的位置，精度为2纳米。这个能力很强的人 这项技术为研究快速单分子动力学打开了新的大门。 在这项提议的第一个目标中，我们构建了一个定制的iSCAT显微镜，并使用它来发现 微管马达使用三磷酸腺苷来产生力量。在第二个目标中，使用iSCAT来跟踪个体的命运 微管格子内的微管蛋白亚基，以便定量描述微管如何动态 在没有和存在抗有丝分裂药物的情况下，不稳定是可以控制的。在第三个目标中，新的进步 分析技术被用来研究微管在有丝分裂过程中如何附着在动点上。在这些方面取得成功 AIMS将定量详细地阐明化疗靶点如何发挥作用，并将推进治疗 通过指导新的抗有丝分裂药物的开发，为广泛的癌症选择。

Many chemotherapy drugs target microtubules in order to inhibit mitosis and stop the proliferation of cancer cells. Some mitotic inhibitors alter microtubule assembly kinetics in order to stall or prevent microtubule growth, while others prevent the attachment of microtubules to the relevant mitotic machinery. However, the fundamental processes of microtubule growth and microtubule attachment to kinetochores remain poorly understood. The goal of this proposal is to elucidate how the molecular targets of mitotic inhibitors operate under normal conditions, and how anti-cancer therapeutics alter their mechanisms. The central approach utilized in this study is to reconstitute the mitotic machinery in vitro using purified recombinant proteins. This bottoms-up approach has the advantages of allowing for direct and complete control over experimental conditions, and of enabling single-molecule measurements to be made using powerful analytical techniques. In this proposal, a new microscopy technique called interferometric scattering microscopy (iSCAT) is refined and applied in order to answer mechanism questions centering around microtubules. iSCAT enables direct visualization of unlabeled microtubules at frame rates up to 50,000 frames per second, and can measure the position of proteins labeled with a 30-nm gold nanoparticle with 2-nm precision. This highly capable technique opens new doors for studying fast single-molecule kinetics. In the first aim of this proposal, a custom iSCAT microscope is constructed and used to discover how microtubule motors use ATP to produce force. In the second aim, iSCAT is used to track the fates of individual tubulin subunits within the microtubule lattice in order to quantitatively describe how microtubule dynamic instability is controlled in the absence and presence of anti-mitotic drugs. In the third aim, new advanced analytical techniques are used to study how microtubules attach to kinetochores during mitosis. Success in these aims will elucidate in quantitative detail how chemotherapy targets function, and will advance the treatment options for a broad array of cancers by guiding the development of new anti-mitotic drugs.