Biochemical analysis of mammalian formin effects on actin dynamics

Formins are members of a conserved family of proteins, present in all eukaryotes, that regulate actin dynamics. Mammals have 15 distinct formin genes. From studies to date, surprising variability between these isoforms has been uncovered. All formins examined have several common effects on actin dynamics in that they: (1) accelerate nucleation rate; (2) alter tilament barbed end elongation/depolymerization rates; and (3) antagonize capping protein. However, the potency of each effect can vary greatly between formins. In addition, a subset of formins binds tightly to filament sides and bundle filaments. Even isoforms that are closely related phylogenetically can display marked differences in their effects on actin. This chapter discusses several methods for examining formin function in vitro. We also discuss pitfalls associated with these assays. As one example, the effect of profilin on formin function is difficult to interpret by "pyrene-actin" polymerization assays commonly used in the field and requires assays that can distinguish between filament nucleation and filament elongation. The regulatory mechanisms for formins are not clear and certainly vary between isoforms. A subset of formins is regulated by Rho GTPases, and the assays described in this chapter have been used for characterization of this regulation.