Single molecule mechanics of myosin motors using optical tweezers at sub-millisecond time resolution

我的研究主要集中在肌动蛋白细胞骨架系统上，无数的运动细胞功能是基于此，从肌肉收缩到内分泌和胞吐，细胞运动，胞质分裂，甚至听觉信号转导。为了了解各种肌球蛋白马达的结构动力学和功能特性，这些马达被组织成细胞中高度专业化的运输或力传感系统，我们需要将单分子技术的时间和空间分辨率的极限推向纳米和微秒范围。这将使最终将单分子力学研究与分子动力学模拟联系起来，并获得对这些细胞纳米机器的分子动力学的真正原子理解成为可能。我们正在使用基于定制光学镊子的单分子荧光和机械技术，使我们能够研究单个马达蛋白质产生的皮牛顿力和纳米位移。我们现在的目标是开发一种时间分辨率在亚毫秒范围内的光镊装置。随着时间分辨率的提高，我们希望能够解决早期的acto-myosin相互作用的事件，包括从弱非特异性到立体特异性强结合状态的过渡。化学机械循环的这些部分至今仍然是虚幻的。在这个项目中，我们将特别关注两类肌球蛋白与有趣的细胞生物学功能;（一）一个古老的肌球蛋白类XXI，唯一的肌球蛋白在人类致病性利什曼原虫寄生虫，寄生虫的生存具有至关重要的作用，和（ii）人类肌球蛋白类IX，这起着至关重要的作用，在迁移细胞中的板状伪足的重组和细胞极化的形态发生。这两种肌球蛋白在医学和基础研究方面的机械性质尚不清楚，以前也没有在单分子水平上进行过研究。

My research focuses on the acto-myosin cytoskeletal system on which a myriad of motile cellular functions is based, ranging from muscle contraction to endo-and exocytosis, cell locomotion, cytokinesis and even signal transduction in hearing. In order to understand the structural dynamics and functional properties of the large variety of myosin motors that are organised into highly specialised transport or force sensing systems in the cell, we need to push the limits of the temporal and spatial resolution of single-molecule technology to the nanometre and microsecond range. This will make it possible to finally connect the single molecule mechanical studies to molecular dynamics simulations and to obtain a truly atomic understanding of the molecular dynamics of these cellular nano-machines. We are using single-molecule fluorescence and mechanical techniques based on custom-built optical tweezers that enable us to study the pico-Newton forces and nano-metre displacements produced by single motor proteins. Our aim is now to develop an optical tweezers apparatus with a time resolution in the sub-millisecond range. With the improved time resolution we hope to resolve the early events of the acto-myosin interaction, including the transition from weak non-specific to stereospecific strong binding states. These parts of the chemo-mechanical cycle have remained illusive to date. In this project we will focus specifically on two classes of myosin with intriguing cell biological functions; (i) an ancient myosin class XXI, the only myosin expressed in the human pathogenic Leishmania parasite which has a vital role for the parasite survival, and (ii) human myosin class IX, which plays a critical role in reorganisations of the lamellipodium in migrating cells and during cell polarisation in morphogenesis. The mechanical properties of these two medically and in terms of basic research important classes of myosin are unclear and they have not been studied at the single molecule level before.