Using microfluidics single filament assay to study formin control of actin assembly

Abstract

Formin is a highly processive motor that offers very unique features to control the elongation of actin filaments. When bound to the filament barbed-end, it enhances the addition of profilin–actin from solution to dramatically accelerate actin assembly. The different aspects of formin activity can be explored using single actin filament assays based on the combination of microfluidics with fluorescence microscopy. This chapter describes methods to conduct single filament experiments and explains how to probe formin renucleation as a case study: purification of the proteins, the design, preparation, and assembly of the flow chamber, and how to specifically anchor formins to the surface.