PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium.

The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. We have identified four of the members of this complex in Dictyostelium and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar(-) mutant resulted in cells resembling their scar(-) parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, we find that pirA(-) mutants contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.