While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor-and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.Cytoskeletal remodelling is a highly dynamic, tightly regulated process which drives the formation of cell protrusions necessary for cell movement. Members of the Rho family of GTPases regulate cytoskeletal dynamics by cycling between inactive GDP-bound and active GTP-bound states. Cdc42 activation results in a localized increase in actin polymerization and the formation of filopodia. Immediately downstream of Cdc42 are p21-activated kinase 1 (PAK1) as well as the Wiskott-Aldrich syndrome protein (WASP) family of proteins, both of which are activated through the binding of GTP Cdc42 via the Cdc42-and Rac-binding domain (CRIB or PBD). PAK1 has been reported to localize to regions of cytoskeletal assembly, and the activated form can induce cytoskeletal remodeling at the leading edge via downstream events such as microtubule growth and stathmin phosphorylation (11, 60). A close correlation has been found between the PAK1 activity state and the baseline invasiveness of human breast cancer cells as well as breast tumor grades (52). Paradoxically, the same work demonstrated that breast carcinoma cells expressing the GTPase binding-deficient H83L, H86L PAK1 mutant exhibited extensive membrane ruffling in the cell periphery. The lamellipodia formation and membrane ruffling observed in the presence of PAK1 were subsequently demonstrated by others to be independent of PAK catalytic activity (13). The relationship between the local level of PAK1 activity and the formation of specific subcellular structures is therefore unclear.The WASP-family proteins and the Arp2/3 complex are important nucleators of new actin filaments in response to signals causing cel...