Biophysics of actin nucleation

肌动蛋白成核因子的数量和种类以及多个成核因子之间的潜在串扰对结构-功能研究构成了巨大的挑战。同时，这种多样性也提供了机会，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。我们建议使用功能和结构良好的特点肌动蛋白结合域工程师新的肌动蛋白成核剂具有明确的属性。两个这样的结构域是存在于许多肌动蛋白调节剂中的WH 2结构域和最近描述的Lifeact肽[2]。通过确定的接头序列融合肌动蛋白结合结构域可以产生基本的肌动蛋白成核剂模块。我们将使用已建立的批量荧光测定法和创新的单分子技术，如光子计数直方图分析，荧光互相关分析（FCCS）和单分子Förster共振能量转移（FRET），对影响肌动蛋白聚合的理化参数和我们的合成成核剂进行仔细的体外表征。这些研究将通过在芽殖酵母中表达各种构建体来补充，芽殖酵母是我们已经建立的肌动蛋白聚合机制研究的细胞模型生物。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，定量描述肌动蛋白成核和聚合。

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.