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

我的研究重点是肌球蛋白-肌动蛋白细胞骨架系统，它是无数运动细胞功能的基础，从肌肉收缩到胞内分泌、细胞运动、细胞分裂甚至听力中的信号转导。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到纳米和微秒范围。这将有可能最终将单分子力学研究与分子动力学模拟联系起来，并获得这些细胞纳米机器的分子动力学的真正原子理解。我们正在使用单分子荧光和基于定制光学镊子的机械技术，使我们能够研究单个马达蛋白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学镊子装置。随着时间分辨率的提高，我们希望解决肌动蛋白相互作用的早期事件，包括从弱非特异性到立体特异性强结合状态的转变。化学-机械循环的这些部分至今仍是虚幻的。在这个项目中，我们将特别关注两类具有有趣细胞生物学功能的肌凝蛋白；(i)一个古老的肌球蛋白XXI类，这是唯一在人类致病性利什曼原虫中表达的肌球蛋白，对寄生虫的生存起着至关重要的作用；（ii）人类肌球蛋白IX类，它在迁移细胞的板层基重组和细胞形态发生的极化过程中起着关键作用。这两类重要的肌球蛋白在医学上和基础研究上的力学性质尚不清楚，以前还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.