Combined optical trapping and single molecule fluorescence to study the force-dependent binding kinetics between filamentous actin and its partners

Abstract

Actin filaments are a major constituent of the cytoskeleton in most eukaryotic cells. They function as a connection between the cell body to the focal adhesions in order to transmit forces into and out of the cell. During the force transduction process, many proteins bind to actin filaments in order to initiate a signaling cascade that reaches the cell nucleus. However, the effects of forces in the binding kinetics between actin filaments and actin binding proteins are unknown. This work proposes an experimental setup to study the force-dependent binding kinetics of such proteins at the single molecule level by using an instrument that combines optical trapping with single molecule fluorescence. The main focus of this work was the design and construction of the experimental equipment. The results show position detection capabilities with a resolution of 5 nm. Also, the trap stiffness recorded was in the order of 0.05 pN/nm. With the combination of position and trap stiffness, the force resolution of the instrument is about 0.25 pN. Also, a photobleaching event for a single dye molecule was recorded, proving the single molecule fluorescence capabilities. In addition, a complete experimental assay is described in order to perform studies on how force application affects the binding of actin and actin binding proteins.