VASP is a processive actin polymerase that requires monomeric actin for barbed end association

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and pro mote assembly of both filopodial and lamellipodial actin networks To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro We observed multiple filament binding modes (1) static side binding, (2) side binding with one dimensional diffusion, and (3) processive barbed end tracking Actin monomers antagonize side binding but promote high affinity (K-d = 9 nM) barbed end attachment In low ionic strength buffers, VASP tetramers are weakly processive (K-off = 0 69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti capping activity Based on our observations, we pro pose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly