Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages

The Yersinia outer surface protein invasin binds to β1 integrins on target cells and has been shown to trigger phagocytic uptake by macrophages. Here, we investigated the role of the actin regulator Wiskott–Aldrich syndrome protein (WASp), its effector the Arp2/3 complex and the Rho-GTPases CDC42Hs, Rac and Rho in invasin/β1 integrin-triggered phagocytosis. During uptake of invasin-coated latex beads, the α5β1 integrin, WASp and the Arp2/3 complex were recruited to the developing actin-rich phagocytic cups in primary human macrophages. Blockage of β1 integrins by specific antibodies, inhibition of Arp2/3 function by microinjection of inhibitors or the use of WASp knockout macrophages inhibited phagocytic cup formation and uptake. Furthermore, microinjection of the dominant negative GTPase mutants N17CDC42Hs, N17Rac or the Rho-specific inhibitor C3-transferase into macrophages greatly attenuated invasin-induced formation of cups. These data suggest that during invasin-triggered phagocytosis β1 integrins activate actin polymerization via CDC42Hs, its effector WASp and the Arp2/3 complex. The contribution of Rac and Rho to phagocytic cup formation also suggests a complex interplay between different Rho GTPases during phagocytosis of pathogens.     

Rho-kinase and myosin-II control phagocytic cup formation during CR,but not FcgammaR,phagocytosis

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.     

Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing αv integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires αvβ5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of αvβ5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated αvβ5 integrin. Both pathways are independently activated and required for clearance phagocytosis.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

A role for cofilin and LIM kinase in Listeria-induced phagocytosis

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.     

Biochemical characterization of distinct regions of SPEC molecules and their role in phagocytosis

Cdc42 signaling pathways play important roles in immune cell polarization and cytoskeletal changes. Although the small Cdc42-binding proteins SPEC1 and SPEC2 play a role in F-actin accumulation in activated T lymphocytes, little is known about their precise activities in other cell types. Here, we mapped the Cdc42-binding activity of SPEC1 to the CRIB sequence and a downstream alpha helical region. Biochemical studies revealed that SPEC1 did not interact with a Rac1 switch-of-function mutant capable of inducing Cdc42-like filopodia, potentially eliminating a role for SPECs in this process. A phosphoinositide-binding region was identified within a basic region N-terminal to the CRIB sequence of SPEC1. Using an anti-SPEC2 antibody, we found that endogenous SPEC2 colocalized with Cdc42 at the phagocytic cup of macrophages internalizing zymosan A particles prior to significant F-actin accumulation. Overexpression studies of the related SPEC1 protein induced marked macrophage contraction and prevented particle binding and phagocytosis. Although a Cdc42-binding mutant of SPEC1 still caused macrophage contraction, mutations within the N-terminal cysteines and phosphoinositide-binding region reversed macrophage contraction but still resulted in impaired phagocytosis. These results identify three distinct structural and functional regions within SPECs and demonstrate their likely role in early contractile events in phagocytosis.     

A Cdc42 Activation Cycle Coordinated by PI 3-Kinase during Fc Receptor-mediated Phagocytosis

Fcγ Receptor (FcR)-mediated phagocytosis by macrophages requires phosphatidylinositol 3-kinase (PI3K) and activation of the Rho-family GTPases Cdc42 and Rac1. Cdc42 is activated at the advancing edge of the phagocytic cup, where actin is concentrated, and is deactivated at the base of the cup. The timing of 3′ phosphoinositide (3′PI) concentration changes in cup membranes suggests a role for 3′PIs in deactivation of Cdc42. This study examined the relationships between PI3K and the patterns of Rho-family GTPase signaling during phagosome formation. Inhibition of PI3K resulted in persistently active Cdc42 and Rac1, but not Rac2, in stalled phagocytic cups. Patterns of 3′PIs and Rho-family GTPase activities during phagocytosis of 5- and 2-μm-diameter microspheres indicated similar underlying mechanisms despite particle size–dependent sensitivities to PI3K inhibition. Expression of constitutively active Cdc42(G12V) increased 3′PI concentrations in plasma membranes and small phagosomes, indicating a role for Cdc42 in PI3K activation. Cdc42(G12V) inhibited phagocytosis at a later stage than inhibition by dominant negative Cdc42(N17). Together, these studies identified a Cdc42 activation cycle organized by PI3K, in which FcR-activated Cdc42 stimulates PI3K and actin polymerization, and the subsequent increase of 3′PIs in cup membranes inactivates Cdc42 to allow actin recycling necessary for phagosome formation.     

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.     

Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.     

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.     

ArhGAP12 plays dual roles in Stabilin-2 mediated efferocytosis: Regulates Rac1 basal activity and spatiotemporally turns off the Rac1 to orchestrate phagosome maturation

The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.     

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process.     

Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization,Macrophage Phagocytosis,and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.     

Abr and Bcr, two homologous Rac GTPase-activating proteins, control multiple cellular functions of murine macrophages

Small GTPases of the Rho family are key regulators of phagocytic leukocyte function. Abr and Bcr are homologous, multidomain proteins. Their C-terminal domain has GTPase-activating protein (GAP) activity that, in vitro, is specific for Rac and Cdc42. To address the in vivo relevance of these entire proteins, of which little is known, the current study examined the effect of the genetic ablation of Abr and Bcr in murine macrophages. The concomitant loss of Abr and Bcr induced multiple alterations of macrophage cellular behavior known to be under the control of Rac. Macrophages lacking both Abr and Bcr exhibited an atypical, elongated morphology that was reproduced by the ectopic expression of GAP domain mutant Abr and Bcr in a macrophage cell line and of constitutively active Rac in primary macrophages. A robust increase in colony-stimulating factor 1 (CSF-1)-directed motility was observed in macrophages deficient for both proteins and, in response to CSF-1 stimulation, Abr and Bcr transiently translocated to the plasma membrane. Phagocytosis of opsonized particles was also increased in macrophages lacking both proteins and correlated with sustained Rac activation. Bcr and Abr GAP mutant proteins localized around phagosomes and induced distinct phagocytic cup formation. These results identify Abr and Bcr as the only GAPs to date that specifically negatively regulate Rac function in vivo in primary macrophages.     

Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

Objective

Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism.

Methods

The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed.

Results

It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively.

Conclusion

Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.     

Inhibition of "self" engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47-although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPalpha localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47-SIRPalpha interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.     

Dissociation of recruitment and activation of the small G-protein Rac during Fcgamma receptor-mediated phagocytosis

Rho-family proteins play a central role in most actin-dependent processes, including the control and maintenance of cell shape, adhesion, motility, and phagocytosis. Activation of these GTP-binding proteins is tightly regulated spatially and temporally; however, very little is known of the mechanisms involved in their recruitment and activation in vivo. Because of its inducible, restricted signaling, phagocytosis offers an ideal physiological system to delineate the pathways linking surface receptors to actin remodeling via Rho GTPases. In this study, we investigated the involvement of early regulators of Fcgamma receptor signaling in Rac recruitment and activation. Using a combination of receptor mutagenesis, cellular, molecular, and pharmacological approaches, we show that Src family and Syk kinases control Rac and Vav function during phagocytosis. Importantly, both the immunoreceptor tyrosine-based activation motif within Fcgamma receptor cytoplasmic domain and Src kinase control the recruitment of Vav and Rac. However, Syk activity is dispensable for Vav and Rac recruitment. Moreover, we show that Rac and Cdc42 activities coordinate F-actin accumulation at nascent phagosomes. Our results provide new insights in the understanding of the spatiotemporal regulation of Rho-family GTPase function, and of Rac in particular, during phagocytosis. We believe they will contribute to a better understanding of more complex cellular processes, such as cell adhesion and migration.     

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.