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

使用干涉散射显微镜 (iSCAT) 对福尔明介导的肌动蛋白组装进行成像

• 批准号: 320340360
• 负责人: Dr. Nikolas Hundt
• 金额: --
• 依托单位: Lehrstuhl für Zelluläre Physiologie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/320340360?language=en
• 关键词: Imaging; formin; mediated; actin; assembly

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.

肌动蛋白细胞骨架对于许多基本细胞功能至关重要，例如细胞迁移、细胞分裂、细胞与细胞接触的建立、胞吐作用和胞吐作用以及囊泡运输。然而，肌动蛋白丝动力学的分子细节尚未完全阐明。缺乏理解的原因之一是标准荧光显微镜技术无法达到跟踪进入和离开丝的单个肌动蛋白亚基所需的动态范围和时空分辨率的组合。该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。该技术能够根据单个分子的质量对其进行检测、成像和定位，而无需任何标记。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亚基添加和丝退火，并首次能够测量相关动力学。此外，我将结合 iSCAT 和单分子荧光显微镜来研究 formins 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福尔明的各个步骤，并将它们与肌动蛋白亚基掺入丝中相关联。一旦建立了福尔明介导的肌动蛋白组装的成像系统，最终目标是研究福尔明是否以及如何协调原肌球蛋白募集至肌动蛋白丝。

项目成果

Direct Single Molecule Observations of the Unique Mechanical State of Human Myosin-6

人肌球蛋白 6 独特机械状态的直接单分子观察

• DOI: 10.1016/j.bpj.2016.11.1438
• 发表时间: 2017
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Takagi Y;Hundt N;Billington N;Andrecka J;Cole D;Fineberg AJ;Katagiri N;Bird JE;Friedman TB;Kukura P;Sellers JR
• 通讯作者: Sellers JR

Visualization of myosin II filament dynamics in remodeling acto-myosin networks with interferometric scattering microscopy

用干涉散射显微镜观察重构肌动球蛋白网络中肌球蛋白 II 丝动力学的可视化

• DOI: 10.1101/199778
• 发表时间: 2017
• 期刊: bioRxiv
• 作者: Koester D;Hundt N;Young G;Fineberg A;Kukura P ;Mayor S
• 通讯作者: Mayor S

Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

使用干涉散射显微镜在单分子水平上无标记观察肌动蛋白成核和聚合

• DOI: 10.1016/j.bpj.2017.11.2108
• 发表时间: 2018
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Hundt N;Tyler A;Young G;Cole D;Fineberg AJ;Andrecka J;Kukura P
• 通讯作者: Kukura P