Signaling mechanisms that regulate actin-based motility processes in the nervous system

Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood.     

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca²⁺ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca²⁺ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.     

Electrotaxis of lung cancer cells in a multiple-electric-field chip

We report a microfluidic cell culture chip that was used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. Electric field (EF) inside the microchamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1–5 and CL1–0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E = 74–375 mV/mm. CL1–5 cells migrated toward the anode while CL1–0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration. Judging from the different temporal responses of the orientation and the migration, it is proposed that the two EF-induced responses may involve different signaling pathways.     

Morphogenetic guidance cues can interact synergistically and hierarchically in steering nerve cell growth

Nerve cell growth is influenced by guiding properties of its substratum. Microfabricated cell culture substrata were used to determine whether rat dorsal root ganglia (DRG) nerve cells could detect and integrate simultaneous model adhesive and topographic guidance cues. Interference reflection microscopy demonstrated strips of surface contact under the marginal zone of growth cones on planar surfaces which were coincident with actin immunostaining at the periphery of the C-domain. Clusters of focal contacts below the growth cone C-domain delineated the track edges on adhesive gratings. Neurite extension was guided most effectively by adhesive gratings of 25-μm period where highly aligned cells were typically bipolar. Nanometric steps and differences in surface texture between the adhesive tracks was detected using atomic force microscopy (AFM). Neurites did not align to 12- to 100-μm pitch grooves which were less than 1 μm deep. The proportion of aligned neurites increased with groove depth. Maximum neurite alignment was seen when 6-μm-deep, 25-μm-wide grooves contained superimposed parallel adhesive tracks of matched pitch. Neurites aligned preferentially to adhesive tracks superimposed orthogonally over shallow grooves (1 μm deep). Primary neurites aligned increasingly to grooves with orthogonal adhesive tracks as their depth increased. These neurites frequently had highly branched terminal arbours aligned to the orthogonal adhesive tracks. We conclude that morphogenetic guidance cues can interact synergistically and hierarchically to steer nerve cell growth.     

Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay.

The p21ras superfamily, involved in diverse processes including cell growth and intracellular trafficking, possesses intrinsic GTPase activity and cycles between GTP-bound active and GDP-bound quiescent states. This intrinsic activity, which results in down-regulation, is accelerated by GTPase activating proteins (GAPs). Other proteins regulating the GDP/GTP cycle include exchange proteins and dissociation inhibitors. The p21s rho, rac, and cdc42Hs constitute a subfamily implicated in cytoskeletal organization. BCR and n-chimaerin are prototypes of a new GAP family for these p21s. To investigate proteins modulating GTP hydrolysis of the three p21s, we developed a novel overlay assay applicable to tissue extracts. Diverse GAPs with different specificities were identified in all rat tissues. Brain contained rac1 GAPs of 45, 50, 85, 100, and 150 kDa. The p50 and p150 GAPs also act on rhoA and cdc42Hs and are ubiquitous, while the p45-GAP, n-chimaerin, is brain- and testis-specific and acts preferentially on rac1; the p100 GAP acts on both rac1 and cdc42Hs and is brain-specific. A new class of p21-interacting proteins was also identified. This diversity, versatility, and tissue specificity of GAPs may be required for fine control of the down-regulation of GTP-bound p21s and the suggested specific downstream effects of individual GAPs, which could involve "cross-talk" between GAPs and p21s.     

Selective control of basolateral membrane protein polarity by cdc42

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase-deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase-deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine-tune their membrane composition in response to extracellular signals during development, migration and in response to injury.     

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.     

A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20.

We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains. Based on these sequences we screened a human placenta library and cloned the full-length cDNA. The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65. Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only. hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active. The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar. hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.     

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.     

Invasive cell migration is initiated by guided growth of long cellular extensions

The migration of border cells during Drosophila melanogaster oogenesis is a simple and powerful system for studying invasive cell migration in vivo. Border cells are somatic cells that delaminate from the follicular epithelium of an egg chamber and invade the germ line cluster. They migrate between the nurse cells to reach the oocyte, using DE-cadherin for adhesion to the substratum. Border cells take approximately 6 h to migrate a distance of 100 microm. The migration is guided by EGFR (epidermal growth factor receptor) and PVR (platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptor). Here, we show that a single long cellular extension (LCE), several cell diameters in length, is formed at the initiation of migration. The LCE may function as a 'pathfinder' in response to guidance cues. LCE growth requires directional guidance signals and specific adhesion to the substratum. Interference with actin-myosin interactions allows continued LCE growth while preventing translocation of the cell bodies. We discuss similarities between LCEs and axons and the use of LCE-like structures as a general mechanism for initiating invasive migration in vivo.     

Electrical fields in wound healing—An overriding signal that directs cell migration

Injury that disrupts an epithelial layer instantaneously generates endogenous electric fields (EFs), which were detected at human skin wounds over 150 years ago. Recent researches combining molecular, genetic and imaging techniques have provided significant insights into cellular and molecular responses to this “unconventional” signal. One unexpected finding is that the EFs play an overriding guidance role in directing cell migration in epithelial wound healing. In experimental models where other directional cues (e.g., contact inhibition release, population pressure etc.) are present, electric fields of physiological strength override them and direct cell migration. The electrotaxis or galvanotaxis is mediated by polarized activation of multiple signaling pathways that include PI3 kinases/Pten, membrane growth factor receptors and integrins. Genetic manipulation of PI3 kinase/Pten (Phosphoinositide 3-kinases/phosphatase and tensin homolog) and integrin β4 demonstrated the importance of those molecules. The electric fields are therefore a fundamental signal that directs cell migration in wound healing. One of the most challenging question is: How do cells sense the very weak electric signals? Clinically, it is highly desirable to develop practical and reliable technologies for wound healing management exploiting the electric signaling.     

Ephrin-B ligands play a dual role in the control of neural crest cell migration

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. Our aim was to determine how neural crest cells that are specified as neurons and glial cells only migrate ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, we asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, we detected the expression of ephrin-B ligands in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, we show that ephrins block dorsolateral migration of early-migrating neural crest cells because when we disrupt the Eph-ephrin interactions by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, we discovered the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridisation, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signalling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signalling to the actin cytoskeleton, potentially involving the activation of Cdc42/Rac family of GTPases. In support of this hypothesis, we show that adhesion of early migratory cells to an ephrin-B-derivatized substratum results in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin.     

Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae

In amphibian gastrulae, scanning electron microscopy (SEM) has shown the presence of a network of extracellular fibrils on the inner aspect of the ectoderm layer, which serves as the substratum for migration by the presumptive mesoderm cells. In vitro experiments have shown that the fibril network promotes attachment and migration by mesoderm cells, and probably guides the migration by contact guidance. Filopodia of the migrating cells showed preferential attachment to the fibrils. Use of a colloidal gold probe for SEM immunocytochemistry has shown that fibrils observed by SEM contain fibronectin, probably as a major component. This provides direct evidence that the extracellular matrix containing fibronectin provides the substratum and guides cell migration in morphogenetic movement.     

Evidence for the guidance of pronephric duct migration by a craniocaudally traveling adhesion gradient

These experiments were undertaken to identify the general nature of the mechanism that guides the migration of the Ambystoma pronephric duct along the ventral edge of the somite file from its anterior origin to the cloaca. Using scanning electron microscopy in conjunction with microsurgery, we have sought to distinguish among such possibilities as chemotaxis, contact guidance, and gradients of adhesiveness to the substratum. A pronephric duct primordium transplanted to the flank of a host ventral to the primary duct migrates dorsocaudally across the flank to fuse with the primary duct. Removal of potential sources of distant attraction does not alter this behavior, nor do migrating secondary ducts follow any visible structures. A variety of transplantation experiments reveal that the guidance information is not only oriented but also directionally polarized and travels caudad as a wave. These results militate against chemotaxis and contact guidance as guiding influences and indicate that the cells of the pronephric duct tip are directed in their migration by local information which passes caudad over the duct's mesodermal substratum as a wave in register with the advancing wave of somite segmentation. We propose that this duct-guiding information may be a traveling gradient of flank mesoderm cell adhesiveness.     

Steered migration and changed morphology of human astrocytes by an applied electric field

Direct current electric field (DC EF) plays a role in influencing the biological behaviors and functions of cells. We hypothesize that human astrocytes (HAs) could also be influenced in EF. Astrocytes, an important type of nerve cells with a high proportion quantitatively, are generally activated and largely decide the brain repair results after brain injury. So far, no electrotaxis study on HAs has been performed. We here obtained HAs derived from brain trauma patients. After purification and identification, HAs were seeded in the EF chamber and recorded in a time-lapse image system. LY294002 and U0126 were then used to probe the role of PI3K or ERK signaling pathway on cellular behaviors. The results showed that HAs could be guided to migrate to the anode in DC EFs, in a voltage-dependent manner. The HAs displayed elongated cell bodies and reoriented perpendicularly to the EF in morphology. When treated with LY294002 or U0126, alternation of parameters such as cellular verticality, track speed, displacement speed, long axis, vertical length and circularity were inhibited partly as expected, while the EF-induced directedness was not terminated even at a high drug dosage which was not consistent with previous electrotaxis studies. In conclusion, applied EFs steered the patient-derived HAs directional migration and changed morphology, in which PI3K and ERK pathways at least partially participate. The characteristics of HAs to EF stimulation may be involved in wound healing and neural regeneration, which could be utilized as a novel treatment strategy in brain injury.     

A self-consistent cell flux expression for simultaneous chemotaxis and contact guidance in tissues

During wound healing, both chemotaxis and contact guidance can contribute to the migration of blood and tissue cells to the wound. In order to understand the wound healing process, we must thus understand how cells respond to both these simultaneous directional cues, which are not necessarily coaligned. Although chemotaxis and contact guidance have been studied individually, the interaction between them has not been addressed. We extend a stochastic cell movement model, developed by Dickinson and Tranquillo (1995) [6] for individual cues, for simultaneous chemotaxis and contact guidance by a two-parameter perturbation analysis in terms of the two associated cues, a chemotactic factor gradient and aligned tissue fibers. We present results from analysis of the first-order perturbation, which includes the cell flux expression heuristically proposed by others, but reveals paradoxical results for other indices of cell movement, such as the mean-squared displacement. We then present second-order perturbation results that resolve these paradoxical results. Finally, we relate these results to a continuum mechanical model developed by Barocas and Tranquillo (1997) [3] that predicts fiber alignment due to cell traction induced tissue contraction. Received: 30 April 1999 / Revised version: 30 October 1999 / Published online: 14 September 2000     

The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin''s rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin''s rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin''s rhoGEF domain. Our results suggest that obscurin''s rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.     

beta4 integrin and epidermal growth factor coordinately regulate electric field-mediated directional migration via Rac1

Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.     

Sprouty regulates cell migration by inhibiting the activation of Rac1 GTPase

Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Rac1, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Rac1(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Rac1(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Rac1(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Rac1 was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Rac1 activation.