Imaging the dynamics of actin-associated proteins in motile cells using ultra-fast, multi-spectral TIRF microscopy

本项目旨在以高空间和时间分辨率研究运动盘状盘骨细胞皮层中肌动蛋白相关蛋白的募集和动态。特别是，我们将重点研究与肌动蛋白动力学相关的蛋白质，包括前缘、行波、斑块和网络的形成。我们的目标是用单分子分辨率可视化参与的蛋白质，如myosin-IB，并期望深入了解它们可能组成多聚体复合物以及它们参与肌动蛋白组织。由于初步实验表明，大多数肌动蛋白斑块之前都有含有网格蛋白的点状结构，因此对网格蛋白动力学的研究将是该项目的第二个主要部分。分析网格蛋白结构的运动性将使我们能够区分质膜附近的各种运动模式，并研究完整细胞中微管依赖的运动活动。使用双色荧光方法同时成像肌动蛋白和网格蛋白组分将允许更好地了解动态肌动蛋白结构参与网格蛋白介导的内吞作用。为了解决这些任务，我们将使用高灵敏度和超快速荧光显微镜相结合；(i)采用宽场全反射荧光（TIRF）照明的低背景荧光成像，（ii）采用电子倍增电荷耦合器件（EMCCD）技术的每秒100帧及以上的超快图像采集，（iii）采用光谱分裂的同时多色成像，以及（iv）采用纳米分辨率的高度平行粒子跟踪的计算机算法。

It is the goal of the project to study the recruitment and the dynamics of actin-associated proteins in the cortex of motile Dictyostelium Discoideum cells with high spatial and temporal resolution. In particular, we will focus our studies on proteins involved in actin dynamics with respect to the formation of leading edges, traveling waves, patches and networks. We aim to visualize the participating proteins, such as myosin-IB, with single molecule resolution and expect to gain insight into their possible organization into multimeric complexes as well as into their involvement in actin organization. Because preliminary experiments indicate the majority of actin patches being preceded by spotty structures containing Clathrin, the study of Clathrin dynamics will be a second major part of the project. Analysis of the motility of the Clathrin structures will enable us to distinguish various modes of movement in the vicinity of the plasma membrane and to study microtubule-dependent motor activities in intact cells. Simultaneous imaging of actin and Clathrin components using a dual-color fluorescence approach will allow for a better understanding of the involvement of dynamic actin structures in Clathrin-mediated endocytosis. To tackle these tasks, we will use highly-sensitive and ultra-fast fluorescence microscopy combining; (i) low background fluorescence imaging by widefiled total-internal reflection fluorescence (TIRF) illumination, (ii) ultrafast image acquisition of 100 frames per second and above by applying electron multiplied charge coupled device (EMCCD) technology, (iii) simultaneous multicolor imaging by spectral splitting and (iv) computerized algorithms for highly-parallel particle tracking with nanometer resolution.