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

肌动蛋白细胞骨架对许多基本的细胞功能至关重要，如细胞迁移、细胞分裂、细胞间接触的建立、细胞内分泌和胞外分泌以及囊泡运输。然而，肌动蛋白丝动力学的分子细节尚未完全阐明。这种缺乏理解的一个原因是，标准的荧光显微镜技术不能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丝。本项目旨在利用干涉散射显微镜（iSCAT）建立一种新的肌动蛋白丝组装成像方法。这项技术能够根据单个分子的质量来检测、成像和定位它们，而不需要任何标记。该方法有望揭示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和丝退火，并首次实现相关动力学的测量。此外，我将结合iSCAT和单分子荧光显微镜来研究形成蛋白mDia1和mDia2对肌动蛋白组装的影响。我的成像方法有望通过荧光显微镜直接揭示肌动蛋白丝末端形成蛋白的各个步骤，并将它们与肌动蛋白亚基进入丝的结合联系起来。一旦形成蛋白介导的肌动蛋白组装成像系统建立，最终目标是研究形成蛋白是否以及如何协调原肌凝蛋白向肌动蛋白丝的募集。

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.