Visualizing and Weighing Kinesin-8 Microtubule Depolymerization with Mass Photometry

微管是细胞骨架的重要组成部分，也是细胞分裂和运动的关键。虽然它们本质上是高度动态的结构，但它们的长度也受其他调节因子的控制，如Kinesin 8和13家族的马达蛋白。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。解聚的动蛋白不运输小泡，但以微管末端为目标进行解聚。虽然它们向微管末端的移位已经被很好地理解了，但它们的水解周期如何与微管蛋白的去除相耦合，以及每次末端遭遇有多少微管蛋白二聚体被去除尚不清楚。在这里，我们将开发一种新型的干涉反射显微镜(IRM)设备，用于(I)对单个萌芽酵母kinesin-8 Kip3进行无标记跟踪，以及(Ii)同时测量其在解聚过程中微管末端的质量去除。KIP3将被用作微管解聚酶的模型系统，用于重组的、含有纯化成分的体外分析。我们的新型IRM装置将通过从蛋白质和样品玻璃表面散射的光的干涉来实现对单个蛋白质的成像。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，IRM显微镜也是一种质量光度计。此外，质心的测量可以实现纳米级精度的单分子定位。在IRM质量光度计原型上的初步测量证实了能够分辨单个微管蛋白和Kip3单体的质量。在商用质量光度计上，原则上可以测量Kip3的微管解聚，但测量时间受光损伤的限制。其他挑战包括动蛋白、微管和微管之间的显著分子量差异，以及比率对比度的表面距离依赖性。为了克服这些挑战，我们将开发和优化一种结合全内反射荧光(TIRF)显微镜的新型IRM质量光度仪器。优化将与计算方法相辅相成，以提高对比度、粒子检测和考虑其表面距离。有了优化的设置，我们将能够在微管上跟踪未标记的马达蛋白质，并测量质量去除或添加到10 kDa的分子质量，与商业系统相比，光减少了大约100-1000倍。后者将允许足够长的观察时间来观察微管解聚而不会造成光损伤。通过解析单个Kip3马达与微管的相互作用和测量去除微管的数量，我们将对解聚机理有更深入的了解。从长远来看，我们希望我们在微管解聚酶方面的工作将有益于癌症研究。

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.