Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.     

Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

Daily alphavbeta5 integrin-dependent phagocytosis of spent photoreceptor outer segment fragments by the retinal pigment epithelium (RPE) is critical for retinal function. This study identifies a key role for focal adhesion kinase (FAK) in RPE phagocytosis. Particle binding increases FAK complex formation with alphavbeta5 receptors at the apical, phagocytic RPE surface and activates FAK. Subsequent particle engulfment coincides with dissociation of activated FAK from alphavbeta5. Mutant FAK retaining focal adhesion targeting but lacking kinase activity interferes with recruitment of full-length FAK to alphavbeta5 and abrogates FAK activation in response to RPE phagocytic challenge. Such inhibition of FAK signaling has no effect on alphavbeta5-dependent binding of particles but blocks their engulfment. Conversely, FAK re-expression promotes particle engulfment by FAK null fibroblasts. Selective ligation of alphavbeta5 receptors at the apical RPE surface is sufficient to phosphorylate and mobilize FAK. Furthermore, FAK phagocytic signaling is independent of the internalization receptor MerTK. In contrast, inhibition of FAK signaling diminishes MerTK phosphorylation. These results demonstrate that FAK provides an essential link between binding and engulfment mechanisms of integrin-mediated 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.     

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.     

Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis

Tubby and tubby-like protein 1 (Tulp1) are newly identified phagocytosis ligands to facilitate retinal pigment epithelium (RPE) and macrophage phagocytosis. Both proteins without classical signal peptide have been demonstrated with unconventional secretion. Here, we characterized them as novel MerTK ligands to facilitate phagocytosis. Tulp1 interacts with Tyro3, Axl and MerTK of the TAM receptor tyrosine kinase subfamily, whereas tubby binds only to MerTK. Excessive soluble MerTK extracellular domain blocked tubby- or Tulp1-mediated phagocytosis. Both ligands induced MerTK activation with receptor phosphorylation and signalling cascade, including non-muscle myosin II redistribution and co-localization with phagosomes. Tubby and Tulp1 are bridging molecules with their N-terminal region as MerTK-binding domain and C-terminal region as phagocytosis prey-binding domain (PPBD). Five minimal phagocytic determinants (MPDs) of K/R(X)(1-2)KKK in Tulp1 N-terminus were defined as essential motifs for MerTK binding, receptor phosphorylation and phagocytosis. PPBD was mapped to the highly conserved 54 amino acids at the C-terminal end of tubby and Tulp1. These data suggest that tubby and Tulp1 are novel bridging molecules to facilitate phagocytosis through MerTK.     

G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca²⁺ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.     

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.     

Endogenous Gas6 and Ca2+ -channel activation modulate phagocytosis by retinal pigment epithelium

Mutation or loss of MerTK as well as deficiency of vβ5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca²⁺-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca²⁺-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca²⁺-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca²⁺-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca²⁺-channels could be regulated downstream of both MerTK and vβ5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.     

A Highlights from MBoC Selection: The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.     

Canonical Wnt/β-Catenin Signalling Is Essential for Optic Cup Formation

A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.     

Creatine Kinase–Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis

Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.     

MERTK Interactions with SH2-Domain Proteins in the Retinal Pigment Epithelium

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK_571–999), purified and phosphorylated. Ni²⁺-NTA pull downs were performed using 6xHis-rMERTK_571–999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK_571–999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.     

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.     

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2^−/− mice the phagocytosis of outer segments was impaired, and in ANX A2^−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2^−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.     

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P₂ to PI(3,4,5)P₃, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P₃ and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.     

Cdc42 Regulates Fcγ Receptor-mediated Phagocytosis through the Activation and Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) and Neural-WASP

Cdc42 is a key regulator of the actin cytoskeleton and activator of Wiskott-Aldrich syndrome protein (WASP). Although several studies have separately demonstrated the requirement for both Cdc42 and WASP in Fc_γ receptor (Fc_γR)-mediated phagocytosis, their precise roles in the signal cascade leading to engulfment are still unclear. Reduction of endogenous Cdc42 expression by using RNA-mediated interference (short hairpin RNA [shRNA]) severely impaired the phagocytic capacity of RAW/LR5 macrophages, due to defects in phagocytic cup formation, actin assembly, and pseudopod extension. Addition of wiskostatin, a WASP/neural-WASP (N-WASP) inhibitor showed extensive inhibition of phagocytosis, actin assembly, and cell extension identical to the phenotype seen upon reduction of Cdc42 expression. However, using WASP-deficient bone marrow-derived macrophages or shRNA of WASP or N-WASP indicated a requirement for both WASP and N-WASP in phagocytosis. Cdc42 was necessary for WASP/N-WASP activation, as determined using a conformation-sensitive antibody against WASP/N-WASP and partial restoration of phagocytosis in Cdc42 reduced cells by expression of a constitutively activated WASP. In addition, Cdc42 was required for proper WASP tyrosine phosphorylation, which was also necessary for phagocytosis. These results indicate that Cdc42 is essential for the activation of WASP and N-WASP, leading to actin assembly and phagocytic cup formation by macrophages during Fc_γR-mediated phagocytosis.     

TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ~190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42^V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42^V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.     

Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gα_i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gα_i proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca²⁺ that was partially sensitive to Gα_i nucleotide exchange inhibition and expression of GTP-bound Gα_i recruited Elmo1 to the plasma membrane. Reducing GDP-Gα_i nucleotide exchange, decreasing Gα_i expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gα_i remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.