Biophysics of actin nucleation

肌动蛋白成核的生物物理学

• 批准号: 170417464
• 负责人: Professor Dr. Don C. Lamb
• 金额: --
• 依托单位: Department Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/170417464?language=en
• 关键词: Biophysics; actin; nucleation

The number and variety of actin nucleation factors coupled with potential crosstalk between multiple nucleators poses a huge challenge to structure-function studies. At the same time this diversity also provides the opportunity to extract fundamental parameters of the actin nucleation process and to follow a synthetic biology approach. We propose to use functionally and structurally well characterized actin binding domains to engineer novel actin nucleators with well-defined properties. Two such domains are the WH2 domain present in many actin regulators and the recently described Lifeact peptide [2]. Fusion of actin binding domains via defined linker sequences could generate a basic actin nucleator module. We will perform careful in vitro characterization of physicochemical parameters influencing actin polymerization and of our synthetic nucleators using established bulk fluorimetric assays and innovative single molecule techniques such as photon counting histogram analysis, Fluorescence Cross-Correlation Analysis (FCCS) and single molecule Förster Resonance Energy Transfer (FRET). These studies will be complemented by expression of the various constructs in budding yeast, which we have established as cellular model organisms for mechanistic studies of actin polymerization. Finally, by controlling physicochemical parameters and binding kinetics of actin nucleators we will be able to formulate improved mathematical models that quantitatively describe actin nucleation and polymerization.

肌动蛋白成核因子的数量和种类以及多个成核因子之间潜在的串扰对结构功能研究提出了巨大的挑战。同时，这种多样性还提供了提取肌动蛋白成核过程的基本参数并遵循合成生物学方法的机会。我们建议使用功能和结构上明确表征的肌动蛋白结合域来设计具有明确特性的新型肌动蛋白成核剂。两个这样的结构域是许多肌动蛋白调节因子中存在的 WH2 结构域和最近描述的 Lifeact 肽 [2]。通过定义的接头序列融合肌动蛋白结合域可以产生基本的肌动蛋白成核模块。我们将使用已建立的批量荧光测定和创新的单分子技术（例如光子计数直方图分析、荧光互相关分析（FCCS）和单分子福斯特共振能量转移（FRET））对影响肌动蛋白聚合的物理化学参数和我们的合成成核剂进行仔细的体外表征。这些研究将通过芽殖酵母中各种构建体的表达得到补充，我们已将其建立为用于肌动蛋白聚合机制研究的细胞模型生物体。最后，通过控制肌动蛋白成核剂的物理化学参数和结合动力学，我们将能够制定改进的数学模型来定量描述肌动蛋白成核和聚合。