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

许多化疗药物靶向微管，以抑制有丝分裂并阻止微管的增殖。 癌细胞一些有丝分裂抑制剂改变微管组装动力学，以阻止或阻止微管 生长，而另一些则阻止微管附着到相关的有丝分裂机器。但 微管生长和微管与动粒附着的基本过程仍然很差 明白这个提议的目的是阐明有丝分裂抑制剂的分子靶点是如何在 正常情况下，以及抗癌治疗如何改变其机制。 本研究中使用的中心方法是使用纯化的重组蛋白在体外重建有丝分裂机制。 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，并使单分子测量能够使用强大的 分析技术。在这个建议中，一种新的显微技术称为干涉散射显微镜 （iSCAT）的完善和应用，以回答围绕微管的机制问题。iSCAT 能够以高达每秒50，000帧的帧速率直接观察未标记的微管，并且可以 以2 nm的精度测量用30 nm金纳米颗粒标记的蛋白质的位置。这个能力很强的 这项技术为研究快速单分子动力学打开了新的大门。 在本提案的第一个目标中，构建了一个定制的iSCAT显微镜，并用于发现 微管马达使用ATP产生力。在第二个目标中，iSCAT用于跟踪个体的命运 微管蛋白亚单位内的微管晶格，以定量描述如何微管动态 在不存在和存在抗有丝分裂药物的情况下控制不稳定性。在第三个目标中，新的先进 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粒上。成功在这些 aims将定量详细阐明化疗靶点如何发挥作用，并将促进治疗 通过指导新的抗有丝分裂药物的开发，为广泛的癌症提供了选择。

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.