Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin. INTRODUCTIONVillin, an epithelial cell-specific protein belongs to a family of actin-binding proteins that contain segments that display internal homology with each other (Arpin et al., 1988). The amino terminal core of villin retains the actin-capping and -severing functions of villin, whereas the carboxyl terminal headpiece enables villin to cross-link actin filaments. The actin-modifying properties of villin are regulated in vitro by calcium (Northrop et al., 1986), phosphoinositides (Janmey and Matsudaira, 1988; and tyrosine phosphorylation . It has been assumed for several years that villin's actin-bundling and not actin-severing functions are important because nonphysiologically high Ca 2ϩ concentrations (200 M) are required to activate villin's actin-severing activity. However, studies done with the villin knockout mice suggest that in the absence of villin, the actin-bundling properties associated with villin can be substituted by other proteins in the microvilli (Pinson et al., 1998); on the other hand, the actin-severing activity of the microvilli is lost (Ferrary et al., 1999). In recent years, we have demonstrated that villin's actin-modifying functions can be regulated in vitro by tyrosine phosphorylation and phosphatidylinositol bisphosphate (PIP 2 ), suggesting that villin has the potential to function as a link between receptor activation and actin cytoskeleton reorganization even in the absence of high calcium (Arora and McCulloch, 1996;Zhai et al., 2001;. In addition, we have recently demonstrated that the autoinhibited conformation of villin can be released by tyrosine phosp...

. RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells.

Reactive oxygen species (ROS) disrupt the barrier function of airway epithelial cells through a mechanism that appears to involve remodeling of the actin cytoskeleton. Similarly, keratinocyte growth factor (KGF) has been shown to protect against ROS-induced loss of barrier function through a mechanism that may also involve the actin cytoskeleton. To further determine the role of the actin cytoskeleton in ROS-induced barrier injury, we quantified the relative amount of total actin associated with the cytoskeleton following exposure to hydrogen peroxide (H(2)O(2)) and pretreatment with KGF. We also determined the role of the actin-myosin contractile mechanism in the process by quantifying the relative amount of myosin heavy chain (MHC) associated with the cytoskeleton. While the transepithelial resistance (TER) of a monolayer of airway epithelial cells (Calu-3) decreased after 2 h of continuous exposure to 0.5 mM H(2)O(2), actin and MHC, both dissociated from the cytoskeleton within 15 min of H(2)O(2) exposure. The TER of the monolayers remained depressed although both actin and myosin returned to the cytoskeleton by 4 h after the initiation of H(2)O(2) exposure. Filamentous actin (f-actin) staining suggested that the re-associating actin took the form of short fibers associated with cortical actin rather than long stress fibers. Furthermore, pretreatment with KGF prevented the loss of actin and MHC from the actin cytoskeleton but did not prevent the decrease in TER. These studies suggest that actin disassembly from the cytoskeleton is important in the loss of barrier function, but that it is not the overall amount of actin that is associated with the cytoskeleton that is important, rather it is the contribution this actin makes to the architectural cohesiveness of the cell that contributes to the barrier function.