A dual role for Rac1 GTPases in the regulation of cell motility

Abstract

SummaryRac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTPbound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.

A dual role model for active Rac1 in cell migration

“…Interestingly, we found that the two Rac1 effectors were enriched at the opposite regions in the cortex of randomly migrating Dictyostelium cells. 35 It is perhaps not that surprising that the two Rac1 effectors localize to different cortical regions in migrating cells. Activated Rac1 proteins interact with numerous proteins implicated in the regulation of actin dynamics and at least a half dozen have been detected in Dictyostelium so far: multiple IQGAP-related proteins, e.g., DGAP1 36 and GAPA, 37 Scar/ WAVE, 38 formins, 39 various PAK-related kinases 40 and WASP.…”

“…These responses are transient and last approximately 30 sec, a time interval which is comparable to the persistence time of a leading pseudopodium during random migration in Dictyostelium. 35 When actin polymerization does not occur, either during spontaneous nonmotile intervals or when it is inhibited by polymerization-blocking agents, the cell cortex remains labeled only with the DGAP1 probe. 35 Thus, it appears that the major fraction of active Rac1 is bound in a quaternary cortical complex that encompasses DGAP1, Rac1-GTP and the heterodimeric actin-bundling proteins cortexillin I and II.…”

“…35 When actin polymerization does not occur, either during spontaneous nonmotile intervals or when it is inhibited by polymerization-blocking agents, the cell cortex remains labeled only with the DGAP1 probe. 35 Thus, it appears that the major fraction of active Rac1 is bound in a quaternary cortical complex that encompasses DGAP1, Rac1-GTP and the heterodimeric actin-bundling proteins cortexillin I and II. 42 In order to initiate local protrusion of a pseudopodium, conversely influence the local concentration of PIP3, the central role is assumed by a pair of effectors, DGAP1/cortexillin and Scar/WAVE complex, that compete for a common pool of active Rac1 (Fig.…”

A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin

“…S2D). This behavior resembled the one of the isolated CRIB motifs expressed as GFP fusions such as the one of PAK1 that localized to leading edges of migrating cells and was used as a probe to monitor the distribution of active Rac (21,22). For comparison, GFP-cor WT was also present in the cytosol and strongly accumulated in new extensions (Fig.…”

“…As coronin is quite an abundant protein, its loss could lead to an enhanced level of GTP-loaded Rac. We probed this directly in vegetative corA − cells by carrying out a pull-down assay with the GST-tagged p21 binding domain of PAK (GST-PBD-i.e., the CRIB domain of PAK) from rat that interacts with activated Racs in cell lysates (21). Precipitated Rac was revealed by probing with polyclonal Rac antibodies.…”