The inhibitory switch (IS) domain of p21-activated kinase 1 (PAK1) stabilizes full-length PAK1 in an inactive conformation by binding to the PAK1 kinase domain. Competitive binding of small GTPases to the IS domain disrupts the autoinhibitory interactions and exposes the IS domain binding site on the surface of the kinase domain. To build an affinity reagent that selectively binds the activated state of PAK1, we used molecular modeling to re-engineer the isolated IS domain so that it was soluble and stable, did not bind to GTPases and bound more tightly to the PAK1 kinase domain. Three design strategies were tested: in the first and second case, extension and redesign of the N-terminus were used to expand the hydrophobic core of the domain and in the third case the termini were redesigned to be adjacent in space so that that the domain could be stabilized by insertion into a loop in a host cyan fluorescent protein (CFP). The best-performing design, called CFP-PAcKer, was based on the third strategy and bound the kinase domain of PAK1 with an affinity of 400 nM. CFP-PAcKer binds more tightly to a full-length variant of PAK1 that is stabilized in the ‘open’ state (Kd = 3.3 µM) than to full-length PAK1 in the ‘closed’ state (undetectable affinity), and binding can be monitored with fluorescence by placing an environmentally sensitive fluorescence dye on CFP-PAcKer adjacent to the binding site.
Computational protein design; Rosetta; merocyanine dye; p21-activated kinase
Cellular signal transduction occurs in complex and redundant interaction networks that are best examined at the level of single cells by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches to determine the relationships between different networks, studied in separate cells.
Structural modifications of previously reported merocyanine dyes (J. Am. Chem. Soc.
125, 4132–4145) were found to greatly enhance the solvent dependence of their absorbance and fluorescence emission maxima. Density functional theory (DFT) calculations have been performed to understand the differences in optical properties between the new and previously synthesized dyes. Absorption and emission energies were calculated for several new dyes using DFT vertical self-consistent reaction field methods (VSCRF). Geometries of ground and excited states were optimized with a Conductor-like screening model (COSMO) and self-consistent-field (SCF) methods. The new dyes have enhanced zwitterionic character in the ground state, and much lower polarity in the excited state, as shown by the DFT-VSCRF calculations. Consistently, the position of the absorption bands are strongly blue-shifted in more polar solvent (methanol compared to benzene) as predicted by the DFT spectral calculations. Inclusion of explicit H-bonding solvent molecules within the quantum model further enhances the predicted shifts, and is consistent with the observed spectral broadening. Smaller, but significant spectral shifts in polar versus nonpolar solvent are predicted and observed for emission bands. The new dyes show large fluorescence quantum yields in polar hydrogen bonding solvents; qualitatively, the longest bonds along the conjugated chain at the excited S1 state minimum are shorter in the more polar solvent, inhibiting photoisomerization. The loss of photostability of the dyes is a consequence of the reaction with and electron transfer to singlet oxygen, starting oxidative dye cleavage. The calculated vertical ionization potentials of three dyes I-SO, AI-SO(4), and AI-BA(4) in benzene and methanol are consistent with their relative photobleaching rates; the charge distributions along the conjugated chains for the three dyes are similarly predictive of higher reaction rates for AI-SO(4) and AI-BA(4) than for I-SO. Time dependent DFT (TDDFT) calculations were also performed on AI-BA(4); these were less accurate than the VSCRF method in predicting the absorption energy shift from benzene (C6H6) to methanol (MeOH).
A simple one-pot-procedure for preparation of protein-reactive, water soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato group directly on the dye fluorophore system, dyes with high fluorescence quantum yields in water were generated. Both iodo- and chloroacetamido- derivatives were shown to be useful in protein labeling. Less reactive chloroacetamides will be preferential for selective labeling of the most reactive cysteines.
Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Ja helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark, but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49-fold and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light dependent co-localization, which we demonstrate with photoactivable gene transcription in yeast.
We present here the phasor approach to biosensor FRET detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement.
Fluorescence lifetime; phasor analysis; Förster resonance energy transfer; biosensor
Many of the more than 20 mammalian proteins with N-BAR domains1-2 control cell architecture3 and endocytosis4-5 by associating with curved sections of the plasma membrane (PM)6. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the PM or indirectly by PM-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally-induced inward deformation of the PM by cone-shaped nanostructures (Nanocones) and internally-induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved PM. Markedly, live-cell imaging in adherent cells showed selective recruitment of full length N-BAR proteins and isolated N-BAR domains to PM sub-regions above Nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by Nanocones. We further showed that N-BAR domains are periodically recruited to curved PM sites during local lamellipodia retraction in the front of migrating cells. Recruitment required Myosin II-generated force applied to PM connected actin cables. Together, our study shows that N-BAR domains can be directly recruited to the PM by external push or internal pull forces that locally curve the PM.
Here we describe a method for the engineered regulation of protein kinases in living cells, the design and application of RapR (rapamycin regulated) kinases. The RapR kinase method enables activation of kinases with high specificity and precise temporal control. Insertion of an engineered allosteric switch, the iFKBP domain, at a structurally conserved position within the kinase catalytic domain makes the modified kinase inactive. Treatment with rapamycin or its non-immunosuppresive analogs triggers interaction with a small FKBP-rapamycin-binding domain (FRB), restoring the activity of the kinase. The reagents used in this method are genetically encoded or membrane permeable, enabling ready application in many systems. Based on the structural similarity of kinase catalytic domains, this method is likely applicable to a wide variety of kinases. Successful regulation has already been demonstrated for three kinases representing both tyrosine and serine/threonine kinase families (p38, FAK, Src). Procedures for designing and testing RapR kinases are discussed.
kinase; allosteric; activation; phosphorylation
Signaling networks in living systems are coordinated through subcellular compartmentalization and precise timing of activation. These spatiotemporal aspects ensure the fidelity of signaling while contributing to the diversity and of downstream events. This is studied through development of molecular tools that generate localized and precisely timed protein activity in living systems. To study the molecular events responsible for cytoskeletal changes in real time, we generated versions of Rho family GTPases whose interactions with downstream effectors is controlled by light. GTPases were grafted to the phototropin LOV (light, oxygen, or voltage) domain (Huala, E., Oeller, P. W., Liscum, E., Han, I., Larsen, E., and Briggs, W. R. (1997). Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science
278, 2120–2123.) via an alpha helix on the LOV C-terminus (Wu, Y. I., Frey, D., Lungu, O. I., Jaehrig, A., Schlichting, I., Kuhlman, B., and Hahn, K. M. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature
461, 104–108.). The LOV domain sterically blocked the GTPase active site until it was irradiated. Exposure to 400–500 nm light caused unwinding of the helix linking the LOV domain to the GTPase, relieving steric inhibition. The change was reversible and repeatable, and the protein could be returned to its inactive state simply by turning off the light. The LOV domain incorporates a flavin as the active chromophore. This naturally occurring molecule is incorporated simply upon expression of the LOV fusion in cells or animals, permitting ready control of GTPase function in different systems. In cultured single cells, light-activated Rac leads to membrane ruffling, protrusion, and migration. In collectively migrating border cells in the Drosophila ovary, focal activation of photoactivatable Rac (PA-Rac) in a single cell is sufficient to redirect the entire group. PA-Rac in a single cell also rescues the phenotype caused by loss of endogenous guidance receptor signaling in the whole group. These findings demonstrate that cells within the border cell cluster communicate and are guided collectively. Here, we describe optimization and application of PA-Rac using detailed examples that we hope will help others apply the approach to different proteins and in a variety of different cells, tissues, and organisms.
We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge.
S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.
Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools.
The disassembly and withdrawal of vimentin intermediate filaments (VIF) from the plasma membrane induces membrane ruffling and the formation of a lamellipodium. Conversely, lamellipodium formation is inhibited when VIF are present.
Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.
Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P3-PI(3,4)P2 during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.
Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity, but does not affect other protein interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases.
Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.
Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na+/H+ exchange. Yet, the functional relationship between Na+/H+ exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na+/H+ exchange, elevating cytosolic pH and stimulating Na+ influx. Remarkably, although inhibition of Na+/H+ exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na+ influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na+/H+ exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na+/H+ exchange.
In this article it is shown that EGF stimulation leads to rapid activation of RhoG through Vav GEFs and the GEF PLEKHG6. Importantly, different cellular responses induced by EGF are determined by the available GEFs. Furthermore, this article presents results showing that EGF-stimulated cell migration and EGFR internalization are regulated by RhoG.
RhoG is a member of the Rac-like subgroup of Rho GTPases and has been linked to a variety of different cellular functions. Nevertheless, many aspects of RhoG upstream and downstream signaling remain unclear; in particular, few extracellular stimuli that modulate RhoG activity have been identified. Here, we describe that stimulation of epithelial cells with epidermal growth factor leads to strong and rapid activation of RhoG. Importantly, this rapid activation was not observed with other growth factors tested. The kinetics of RhoG activation after epidermal growth factor (EGF) stimulation parallel the previously described Rac1 activation. However, we show that both GTPases are activated independently of one another. Kinase inhibition studies indicate that the rapid activation of RhoG and Rac1 after EGF treatment requires the activity of the EGF receptor kinase, but neither phosphatidylinositol 3-kinase nor Src kinases. By using nucleotide-free RhoG pull-down assays and small interfering RNA-mediated knockdown studies, we further show that guanine-nucleotide exchange factors (GEFs) of the Vav family mediate EGF-induced rapid activation of RhoG. In addition, we found that in certain cell types the recently described RhoG GEF PLEKHG6 can also contribute to the rapid activation of RhoG after EGF stimulation. Finally, we present results that show that RhoG has functions in EGF-stimulated cell migration and in regulating EGF receptor internalization.
The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics1-3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4-7 and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac18, and Rac1 and RhoA are mutually inhibitory9-12. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination both through simultaneous visualization of two GTPase biosensors and using a “computational multiplexing” approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.
Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-κB, C/EBP and serum response factor. The multifaceted host cell activation triggered by Toll-like receptors (TLRs) in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-κB activation, while RhoA was dispensable for type I interferon generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src initiated pathways.
signal transduction; bacterial; viral; transcription factors; cell activation
The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties1 or using photoreactive small molecule ligands2. However, this requires use of toxic UV wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (i.e. through microinjection). We have developed a new approach to produce genetically-encoded photo-activatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics3,4. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin5,6, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, while PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicron precision7,8. Their mutual regulation remains controversial9, with data indicating that Rac inhibits and/or activates Rho10,11. Rac was shown to inhibit RhoA in living cells, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.
Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
GTPase activating protein; Rho GTPases; tumor suppressor
Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.
RhoA; ROCK; Rac; EGF; metastasis