Visualizing and Weighing Kinesin-8 Microtubule Depolymerization with Mass Photometry

使用质量光度法可视化和称量 Kinesin-8 微管解聚

• 批准号: 537583262
• 负责人: Dr. Anita Jannasch
• 金额: --
• 依托单位: Zentrum für Molekularbiologie der Pflanzen (ZMBP)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/537583262?language=en
• 关键词: Visualizing; Weighing; Kinesin; Microtubule; Depolymerization

Microtubules are an essential cytoskeletal component and key for cell division, and motility. While intrinsically they are highly dynamic structures, their length is also controlled by other regulators such as motor proteins of the kinesin 8 and 13 family. Since microtubule length regulation is important for mitosis, these motors have been associated with diseases such as cancer. Depolymerizing kinesins do not transport vesicles, but target microtubule ends for their disassembly. While their translocation to the microtubule end is reasonably well understood, how their hydrolysis cycle is coupled to tubulin removal and how many tubulin dimers are removed per end encounter is unclear. Here, we will develop a novel interference reflection microscopy (IRM) setup for (i) label-free tracking of single budding yeast kinesin-8 Kip3 and (ii) simultaneously measuring its mass removal at microtubule ends during depolymerization. Kip3 will be used as a model system for microtubule depolymerases in reconstituted, in vitro assays with purified components. Our novel IRM setup will enable the imaging of single proteins through interference of light scattered from the protein and the sample glass surface. A rolling differential averaging procedure allows us to determine its ratiometric contrast. Since this contrast is directly proportional to the protein’s molecular weight, the IRM microscope is also a mass photometer. Furthermore, the measurement of the center-of-mass allows single-molecule localization with nanometer precision. Preliminary measurements on an IRM mass photometer prototype confirmed the ability to resolve the mass of single tubulin and Kip3 monomers. On a commercial mass photometer microtubule depolymerization by Kip3 could in principle be measured, but the measurement time was limited by photodamage. Other challenges include the significant molecular weight difference between kinesins, tubulins, and microtubules and the surface-distance dependence of the ratiometric contrast. To overcome these challenges, we will develop and optimize a new IRM mass photometry instrument combined with total internal reflection fluorescence (TIRF) microscopy. The optimization will be complemented with computational approaches to improve contrast, particle detection, and account for its surface distance. With the optimized setup, we will be able to track unlabeled motor proteins on microtubules and measure mass removal or addition down to a molecular weight of 10 kDa with about 100-1000 times less light compared to the commercial system. The latter aspect will allow sufficiently long observation times of microtubule depolymerization without photodamage. By resolving the interaction of single Kip3 motors with microtubules and measuring the number of removed tubulins, we will gain insights into the depolymerization mechanism. In the long term, we hope that our work on mictotubule depolymerases will be beneficial for cancer research.

微管是细胞骨架的重要组成部分，是细胞分裂和运动的关键。虽然本质上它们是高度动态的结构，但它们的长度也受其他调节因子如驱动蛋白8和13家族的马达蛋白控制。由于微管长度调节对有丝分裂很重要，这些马达与癌症等疾病有关。解聚驱动蛋白不运输囊泡，而是靶向微管末端进行分解。虽然他们的易位到微管末端是合理的理解，他们的水解循环是如何耦合到微管蛋白去除和有多少微管蛋白二聚体被删除每个终端遇到是不清楚的。 在这里，我们将开发一种新的干涉反射显微镜（cirp）设置（i）无标记跟踪单个芽殖酵母驱动蛋白-8 Kip 3和（ii）同时测量其质量去除在微管末端解聚过程中。Kip 3将在使用纯化组分的重构体外测定中用作微管解聚酶的模型系统。 我们的新的荧光显微镜设置将使单个蛋白质的成像通过从蛋白质和样品玻璃表面散射的光的干涉。滚动微分平均程序使我们能够确定其比率对比度。由于这种对比度与蛋白质的分子量成正比，因此电子显微镜也是一种质量光度计。此外，质心的测量允许以纳米精度进行单分子定位。对IRM质量光度计原型的初步测量证实了解析单个微管蛋白和Kip 3单体质量的能力。在商业质量光度计上，原则上可以测量由Kip 3引起的微管解聚，但是测量时间受到光损伤的限制。其他挑战包括驱动蛋白、微管蛋白和微管之间的显著分子量差异以及比率对比的表面距离依赖性。为了克服这些挑战，我们将开发和优化一种新的全内反射荧光（TIRF）显微镜相结合的质谱光度计。优化将与计算方法相结合，以提高对比度，颗粒检测，并考虑其表面距离。通过优化的设置，我们将能够跟踪微管上未标记的马达蛋白，并测量分子量低至10 kDa的质量去除或添加，与商业系统相比，光减少约100-1000倍。后一个方面将允许足够长的观察时间的微管解聚没有光损伤。通过解析单个Kip 3马达与微管的相互作用，并测量去除的微管蛋白的数量，我们将深入了解解聚机制。从长远来看，我们希望我们对微管解聚酶的研究将有利于癌症研究。