Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42-and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.Ras superfamily G proteins function as molecular switches in a variety of signaling cascades (51). Among them, Rho family G proteins, including Rho, Rac, and Cdc42, are involved in the regulation of a variety of cellular processes, probably through actin cytoskeleton reorganization (1, 9, 13, 48). In a pioneering work by Nobes and Hall, it was shown that Rho regulates the assembly of the actin stress fiber, that Rac induces lamellipodia and membrane ruffles, and that Cdc42 triggers filopodium formation (41).Rho family G proteins are regulated by three classes of protein, guanine nucleotide exchange factor (GEF), GTPaseactivating protein (GAP), and guanine nucleotide dissociation inhibitor (GDI) (51). GEF promotes the exchange of GDP with GTP, which results in the binding of the G proteins to their effector proteins. A typical GEF protein of the Rho family of G proteins consists of a Dbl homology (DH) domain, which exhibits GEF activity, and additional domains that mediate interactions with peptides or lipids. DOCK180, originally isolated as a protein bound to adapter protein Crk (14), also promotes guanine nucleotide exchange of Rac, although it does not contain the DH domain (20). The GTP on the activated Rho family G protein is hydrolyzed in the presence of GAP to resume the GDP-bound inactive state. GDI not only competes with GEF but also holds the Rho family G proteins in the cytoplasm (43). Therefore, the dissociation of GDI is a prerequisite for the membrane association and activation of the Rho family G prot...

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)–based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582–6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.

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Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Fö rster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP 3 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells. INTRODUCTIONCell migration is an important event during early development, inflammatory responses to infection, and wound healing, and it is an important pathological event during tumor invasion and metastasis. Despite morphological and functional differences, different migratory cells share a conserved set of polarity signals. Kinases for phosphoinositides (PtdInss), Rho GTPases, and the actin and microtubule cytoskeletons play key roles in signaling polarity in cells ranging from Dictyostelium discoideum (Charest and Firtel, 2006) to neurons (Luo, 2000;Aoki et al., 2007) and neutrophils (Van Keymeulen et al., 2006;Wong et al., 2006).PtdInss are a family of phospholipids containing myoinositol as their head group (reviewed in Takenawa and Itoh, 2001). Despite a relatively low abundance in biological membranes, PtdInss have been reported to regulate a myriad of cellular processes. Among them, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] is the major PtdInss at the inner leaflet of the plasma membrane. On growth factor stimulation, intracellular second messengers such as inositol (1,4,5)-trisphosphate (IP 3 ), diacylglycerol (DAG), and phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) are generated from PI(4,5)P 2 .The discovery that the pleckstrin homology (PH) domain in the signaling proteins recognizes specific phosphoinositides revealed that stimulated proteins translocate to specific regions of the membrane to participate in signaling events via interaction with these lipids (reviewed in Di Paolo and De Camilli, 2006). A series of experiments indicated that the PH domains from different proteins recognize different PtdInss and led to the development of a novel t...

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P 2 ], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P 3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P 2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P 2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion. INTRODUCTIONClass I phosphoinositide 3-kinase (PI3K) is a key mediator of intracellular signaling pathways that regulate actin cytoskeletal reorganization and polarized cell migration. Activated PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] to generate phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which, in turn, activates a variety of pleckstrin homology (PH) domain-containing proteins such as Akt (also called protein kinase B) (Saltiel and Pessin, 2002;Sulis and Parsons, 2003). PtdIns(3,4,5)P 3 is dephosphorylated to yield PtdIns(4,5)P 2 by a tumor suppressor protein, phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which is frequently mutated or deleted in various human cancers (Sulis and Parsons, 2003). The resulting PTEN deficiency causes accumulation of PtdIns(3,4,5)P 3 in cells, and, as a consequence, an increase in cell motility in fibroblasts (Liliental et al., 2000;Higuchi et al., 2001;Hafizi et al., 2005). Another dephosphorylated derivative of PtdIns (3,4,5)P 3 is PtdIns(3,4)P 2 produced by phosphoinositide 5-phosphatases, including Src homology 2-containing inositol-5-phosphatase (SHIP). This PtdIns(3,4)P 2 also binds to various PH domain-containing proteins, which overlap significantly to those bound to PtdIns(3,4,5)P 3 (Lemmon and Ferguson, 2000;Maffucci and Falasca, 2001).Among many PH domain-containing proteins, a serinethreonine kinase, Akt, has been studied most extensively. Akt has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (Brazil et al., 2004;Fayard et al., 2005)...

Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (Wnt-PCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by ΔN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncell-autonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.

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