Project Summary/Abstract Dynamic arrays of actin filaments and myosin-2 (“actomyosin”) drive a broad variety of fundamental biological processes including cell division, cell locomotion and wound repair. Such arrays are controlled by the Rho GTPases, proteins that exert their effects on actomyosin by stimulating “effector” proteins when in their active (GTP-bound) state. Traditionally, information flow from the Rho GTPases to the cytoskeleton has been viewed as linear, with GTPase activators (GEFs) stimulating a given GTPase, which then activates effectors which, in turn, modify actomyosin. Subsequently, the process is terminated by inactivation of GTPases by inhibitor proteins (GAPs). However, it is becoming apparent that control of actomyosin arrays entails rapid flux of GTPases from the active to inactive stages that is somehow subject to continual modulation via feedback from the actomyosin itself. A particularly clear example of this behavior is provided by repair of Xenopus oocytes in which circular waves of Rho GTPase activity form around wounds and close inward, as a consequence of spatially biased flux through the GTPase cycle: Rho is preferentially activated at the leading edge of its wave (also known as a zone) and preferentially inactivated at the trailing edge. Further, this spatial bias is somehow linked to the ring of dynamic F-actin that encircles the Rho zone. Here I will test a feedback model in which waves of Rho activity that direct cell wound repair are driven forward by a self-organizing “enzymatic corral” that forms at their trailing edge. Specifically, I will test the hypotheses that 1) trailing edge Rho inactivation depends on an F-actin binding protein known as cortactin; 2) cortactin exerts its effects on Rho activity by serving as a binding site for two GAPs (RG1 and RG8); 3) F-actin, cortactin, RG1 and RG8 are organized into a higher-order dynamic corral, the formation of which is ultimately controlled by the Rho GTPases themselves.

Project Narrative Rho GTPases mediate a multitude of dynamic processes based on actin filaments (F-actin) and myosin-2 (actomyosin) such as cell division, cell migration, and wound healing by stimulating proteins that modulate actomyosin. While information flow from the Rho GTPases to actin filaments and myosin-2 has traditionally been thought to be unidirectional, a growing body of evidence suggests that actomyosin somehow feeds back on Rho GTPase activity and that this feedback is essential. Here I propose a model of Rho GTPase regulation wherein F-actin serves as both a GTPase target and a Rho GTPase regulator by forming the basis of ring-like "enzymatic corral" which sculpts and steers Rho activity by locally inactivating it.