Biophysics of actin nucleation

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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.