Imaging of formin-mediated actin assembly using interferometric scattering microscopy (iSCAT)

肌动蛋白细胞骨架对于许多基本的细胞功能是必不可少的，例如细胞迁移、细胞分裂、细胞间接触的建立、胞内和胞吐以及囊泡运输。然而，肌动蛋白丝动力学的分子细节尚未完全阐明。缺乏理解的一个原因是，标准的荧光显微镜技术没有达到所需的动态范围和时空分辨率的组合，以跟踪单个肌动蛋白亚基进入和离开细丝。该项目的目的是建立一个新的成像方法肌动蛋白丝组装使用干涉散射显微镜（iSCAT）。该技术能够基于单个分子的质量来检测、成像和定位单个分子，而不需要任何标记。该方法有望阐明肌动蛋白丝组装的精确机制，因为它将能够区分单个亚基添加与丝退火，并首次测量相关动力学。此外，我将联合收割机iSCAT与单分子荧光显微镜相结合，以研究形成蛋白mDia1和mDia2对肌动蛋白组装的影响。我的成像方法承诺直接揭示的个别步骤的形成在肌动蛋白丝末端的荧光显微镜，并将它们与肌动蛋白亚基纳入细丝。一旦成像系统的formin介导的肌动蛋白组装建立，最终的目标是调查，是否以及如何formins协调原肌球蛋白招聘肌动蛋白丝。

The actin cytoskeleton is essential for many fundamental cellular functions, such as cell migration, cell division, establishment of cell-cell contacts, endo- and exocytosis and vesicle transport. However, the molecular details underlying actin filament dynamics are not yet fully elucidated. One reason for this lack of understanding is the fact that standard fluorescence microscopy techniques do not reach the required combination of dynamic range and spatio-temporal resolution to track individual actin subunits entering and leaving the filament. The proposed project aims to set up a new imaging approach for actin filament assembly using interferometric scattering microscopy (iSCAT). This technique is capable of detecting, imaging and localising single molecules based on their mass without the necessity of any labels. The method promises to shed light onto the precise mechanisms of actin filament assembly, as it will be able to distinguish single subunit addition from filament annealing and enable the measurement of the associated kinetics for the first time. Furthermore, I will combine iSCAT with single molecule fluorescence microscopy to investigate the impact of the formins mDia1 and mDia2 on actin assembly. My imaging approach promises to directly reveal the individual steps of formins at the tip of actin filament ends by fluorescence microscopy and correlate them with the incorporation of actin subunits into filaments. Once the imaging system for formin-mediated actin assembly is established, the ultimate goal is to investigate, whether and how formins coordinate tropomyosin recruitment to actin filaments.