Cell motility is locally achieved by the interplay between lamellipodia and filopodia at the protrusion sites. The actin cytoskeleton rearranges from a highly branched short filamentous network to well aligned elongated bundles from lamellipodia to filopodia, respectively. This process is governed predominantly by actin binding proteins, VASP and fascin, at the leading edge of migratory cells. Here we use an Arp2/3-complex dependent bead motility assay to study the effect of fascin both on its own and in the presence of VASP. The Young's modulus of phalloidin stabilized comets grown in the presence of fascin increased linearly with concentration above a 0.5 mu M threshold. Inversely, the initial velocity of the comets decreased linearly with fascin concentration above the same threshold. Interestingly, VASP and fascin together increased the Young's modulus of the comets compared to those grown in the presence of only one of the two proteins. This effect was most remarkable at low concentration, below 0.5 and 0.15 mu M for fascin and VASP, respectively. Our results showed that fascin and VASP work cooperatively to enhance the Young's modulus of the actin network within the comets. (C) 2011 Elsevier Inc. All rights reserved.