Effect of Cdc42 domains on filopodia sensing, cell orientation, and haptotaxis

Abstract

Filopodia are sensors which, along with microtubules, regulate the persistence of locomotion. To determine whether protrusions were involved in sensing adhesion, epithelial cells were cultured on platinum and tantalum gradients. Protrusions were defined by an unbiased statistical method of classification as factors 4 (filopodia), 5 (mass distribution), and 7 (nascent neurites). When the prevalence of protrusions was measured in zones of high (H), middle (M), and low (L) adhesiveness, the main differences were in factor 4. Its values were highest at H and declined at M and L regardless of the gradient composition. The significance of the differences was enhanced when T (top/adhesive end) and B (bottom/nonadhesive end) sides of cells were analyzed separately. Since information about sidedness increased the statistical power of the test, this result suggested that cells pointed more filopodia toward the adhesive end. Trends occurred in factors 5 and 7 only when conditions allowed for a marked trend in factor 4. The data showed that gradient sensing is proportional to the prevalence of filopodia, and filopodia are the only protrusions engaged in comparing adhesiveness across a cell. The probability (P) of the significance of a trend was then used to determine how cells sense the gradient. Binding peptides (BPs) were introduced representing sequences critical for Cdc42 docking on a specific partner. BPs for IQGAP (IQ(calmodulin-binding domain)-containing GTPase-activating protein) and ACK (Cdc42-associated kinase) reduced factor 4 values and prevented cell orientation on the gradient. Micrographs showed attenuated or stubby filopodia. These effectors may be implicated in gradient sensing. Another IQGAP BP increased filopodia prevalence and enhanced orientation on the gradient (P<0.00015). A Wiskott–Aldrich syndrome protein (WASP) BP had no effect. When sensing and orientation were abolished, they both failed at the level of filopodia, indicating that filopodia are both sensors and implementers of signals transduced by adhesion.