The actin cytoskeleton forms a major component of all eukaryotic cells. It forms several important intracellular structures and is involved in a large number of crucial functions, for which it works in concert with over 450 actin binding proteins (ABPs). The protein tropomyosin is a key regulator of actin dynamics, since it regulates access of other ABPs to actin. Tropomyosin molecules are alpha-helical coiled coil dimers which interact with each other in a head-to-tail fashion, forming a chain along the actin filament1. In mammals, there are more than 40 isoforms of tropomyosin that show spatial and temporal regulation2. Despite five decades of studies on tropomyosins, detailed kinetics of actin-tropomyosin interactions have not been resolved to date.
We have developed an in vitro reconstitution system for TIRF imaging of actin-tropomyosin interactions at the single filament level. Using microfluidics, the actin filaments are grown on the surface and we then observe the binding of fluorescently labelled tropomyosins in real-time. Thus, using single or two colour imaging, we are able to compare the binding and dynamics of different isoforms at the single filament level, and obtain various interaction parameters for each filament for different isoforms. Furthermore, we are also able to investigate the competition between different isoforms to bind to actin. Insight into whether different isoforms can co-localize on the same filament or whether some completely outcompete others and the kinetics of these processes can reveal some of the mechanisms that drive assembly of actin-tropomyosin filaments in the cell.