Spatially controlled actin filament assembly is critical for numerous proce
sses, including the vectorial cell migration required for wound healing, ce
ll-mediated immunity, and embryogenesis. One protein implicated in the regu
lation of actin assembly is zyxin, a protein concentrated at sites where th
e fast growing ends of actin filaments are enriched. To evaluate the role o
f zyxin in vivo, we developed a specific peptide inhibitor of zyxin functio
n that blocks its interaction with alpha-actinin and displaces it from its
normal subcellular location. Mislocalization of zyxin perturbs cell migrati
on and spreading, and affects the behavior of the cell edge, a structure ma
intained by assembly of actin at sites proximal to the plasma membrane. The
se results support a role for zyxin in cell motility, and demonstrate that
the correct positioning of zyxin within the cell is critical for its physio
logical function. Interestingly, the mislocalization of zyxin in the peptid
e-injected cells is accompanied by disturbances in the distribution of Ena/
VASP family members, proteins that have a well-established role in promotin
g actin assembly. In concert with previous work, our findings suggest that
zyxin promotes the spatially restricted assembly of protein complexes neces
sary for cell motility.