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

阐明有丝分裂抑制剂靶点分子机制的生物物理学方法

基本信息

批准号：
9752984
负责人：
Keith Joseph Mickolajczyk
金额：
$ 9.47万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-13 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9752984
关键词：
ATP Hydrolysis Address Affect Antimitotic Agents Automobile Driving Behavior Biological Biological Assay Biophysics Cell Separation Cell division Cell physiology Cells Cellular Assay Cellular biology Chemicals Complement Complex Custom Development Drug Targeting Family Fluorescence Microscopy Future Geometry Goals Gold Growth Head Heart Image Imagery In Vitro Individual Kinesin Kinetics Kinetochores Label Lateral Lead Learning Malignant Neoplasms Maps Measurement Measures Mechanics Microscope Microscopy Microtubule Polymerization Microtubule Stabilization Microtubules Mission Mitosis Mitotic Mitotic Chromosome Mitotic spindle Molecular Molecular Target Motor Movement National Cancer Institute Paclitaxel Pharmaceutical Preparations Phase Physiology Positioning Attribute Postdoctoral Fellow Process Production Protein Isoforms Proteins Recombinant Proteins Recombinants Research Resolution Speed Structure Techniques Testing Time Travel Tubulin Universities Vinblastine Walking Work anti-cancer therapeutic anticancer research biophysical techniques biophysical tools cancer cell career chemotherapeutic agent chemotherapy chromosome movement design driving force drug testing effective therapy in vitro Assay instrumentation millisecond nanoGold nanometer optical traps physical property prevent professor reconstitution single molecule spatiotemporal success tumor growth working group

项目摘要

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.

许多化疗药物以微管为靶点，以抑制有丝分裂并阻止细胞增殖。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上。然而，微管生长和微管附着到动粒的基本过程仍然很差明白了。该提案的目的是阐明有丝分裂抑制剂的分子靶标如何在有丝分裂抑制剂的作用下发挥作用。正常情况下，以及抗癌疗法如何改变其机制。本研究中使用的中心方法是使用纯化的体外重建有丝分裂机器。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，并能够使用强大的功能进行单分子测量分析技术。在该提案中，一种称为干涉散射显微镜的新显微镜技术 (iSCAT) 被完善和应用，以回答围绕微管的机制问题。 iSCAT 能够以高达每秒 50,000 帧的帧速率直接可视化未标记的微管，并且可以以 2 nm 精度测量用 30 nm 金纳米粒子标记的蛋白质的位置。这位能力极强的该技术为研究快速单分子动力学打开了新的大门。在该提案的第一个目标中，构建了一个定制的 iSCAT 显微镜并用于发现如何微管马达利用 ATP 产生力。第二个目标是使用 iSCAT 来追踪个人的命运微管晶格内的微管蛋白亚基，以定量描述微管动态不稳定性在抗有丝分裂药物的存在和不存在下得到控制。第三个目标，新先进分析技术用于研究有丝分裂过程中微管如何附着到着丝粒上。在这些方面取得成功目标将定量详细地阐明化疗如何靶向发挥作用，并将推进治疗通过指导新的抗有丝分裂药物的开发，为多种癌症提供选择。