The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1

Cell motility requires extensions of the plasma membrane driven by reorganization of the actin cytoskeleton. Small GTPases, particularly the Rho family, are key regulators of this process. A second class of GTPases, the ADP-ribosylation factors (ARFs), have also been implicated in the regulation of the actin cytoskeleton and motility. ARF6 is intimately involved in the regulation of Rac activity; however, the mechanisms by which ARF activation leads to activation of Rac remain poorly understood. We have previously shown that expression of the ARF-GEF ARNO in MDCK cells induces robust activation of Rac, the formation of large lamellipodia, and the onset of motility. We report here that ARNO-dependent activation of Rac is mediated by a bipartite Rac GEF, the Dock180/Elmo complex. Both DOCK180 and Elmo colocalize extensively with ARNO in migrating MDCK cells. Importantly, both a catalytically inactive Dock180 mutant and an Elmo mutant that fails to couple to Dock180 block ARNO-induced Rac activation and motility. In contrast, a similar mutant of the Rac GEF beta-PIX fails to inhibit ARNO-induced Rac activation or motility. Together, these data suggest that ARNO and ARF6 coordinate with the Dock180/Elmo complex to promote Rac activation at the leading edge of migrating cells.     

TLR4 activation induces inflammatory vascular permeability via Dock1 targeting and NOX4 upregulation

The loss of vascular integrity is a cardinal feature of acute inflammatory responses evoked by activation of the TLR4 inflammatory cascade. Utilizing in vitro and in vivo models of inflammatory lung injury, we explored TLR4-mediated dysregulated signaling that results in the loss of endothelial cell (EC) barrier integrity and vascular permeability, focusing on Dock1 and Elmo1 complexes that are intimately involved in regulation of Rac1 GTPase activity, a well recognized modulator of vascular integrity. Marked reductions in Dock1 and Elmo1 expression was observed in lung tissues (porcine, rat, mouse) exposed to TLR4 ligand-mediated acute inflammatory lung injury (LPS, eNAMPT) in combination with injurious mechanical ventilation. Lung tissue levels of Dock1 and Elmo1 were preserved in animals receiving an eNAMPT-neutralizing mAb in conjunction with highly significant decreases in alveolar edema and lung injury severity, consistent with Dock1/Elmo1 as pathologic TLR4 targets directly involved in inflammation-mediated loss of vascular barrier integrity. In vitro studies determined that pharmacologic inhibition of Dock1-mediated activation of Rac1 (TBOPP) significantly exacerbated TLR4 agonist-induced EC barrier dysfunction (LPS, eNAMPT) and attenuated increases in EC barrier integrity elicited by barrier-enhancing ligands of the S1P1 receptor (sphingosine-1-phosphate, Tysiponate). The EC barrier-disrupting influence of Dock1 inhibition on S1PR1 barrier regulation occurred in concert with: 1) suppressed formation of EC barrier-enhancing lamellipodia, 2) altered nmMLCK-mediated MLC2 phosphorylation, and 3) upregulation of NOX4 expression and increased ROS. These studies indicate that Dock1 is essential for maintaining EC junctional integrity and is a critical target in TLR4-mediated inflammatory lung injury.     

The RhoG/ELMO1/Dock180 signaling module is required for spine morphogenesis in hippocampal neurons

Dendritic spines are actin-rich structures, the formation and plasticity of which are regulated by the Rho GTPases in response to synaptic input. Although several guanine nucleotide exchange factors (GEFs) have been implicated in spine development and plasticity in hippocampal neurons, it is not known how many different Rho GEFs contribute to spine morphogenesis or how they coordinate the initiation, establishment, and maintenance of spines. In this study, we screened 70 rat Rho GEFs in cultured hippocampal neurons by RNA interference and identified a number of candidates that affected spine morphogenesis. Of these, Dock180, which plays a pivotal role in a variety of cellular processes including cell migration and phagocytosis, was further investigated. We show that depletion of Dock180 inhibits spine morphogenesis, whereas overexpression of Dock180 promotes spine morphogenesis. ELMO1, a protein necessary for in vivo functions of Dock180, functions in a complex with Dock180 in spine morphogenesis through activating the Rac GTPase. Moreover, RhoG, which functions upstream of the ELMO1/Dock180 complex, is also important for spine formation. Together, our findings uncover a role for the RhoG/ELMO1/Dock180 signaling module in spine morphogenesis in hippocampal neurons.     

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.     

Comparative gene expression analysis of the engulfment and cell motility (ELMO) protein family in the mouse brain

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.     

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.     

Arhgef16, a novel Elmo1 binding partner,promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation

Elmo is an evolutionarily conserved mammalian ortholog of Caenorhabditis elegans CED-12 with proposed roles during the removal of apoptotic cells, cell migration, neurite outgrowth, and myoblast fusion (Katoh and Negishi (2003) [1], Park and Tosello (2007) [2], Grimsley et al. (2004) [3], Hamoud et al. (2014) [4]). Elmo mediates these cellular processes by interacting with various proteins located in the plasma membrane, cytoplasm and nucleus, and by modulating their activities although it has no intrinsic catalytic activity (Park and Tosello (2007) [2], Hamoud et al. (2014) [4], Li et al. (2013) [5], Margaron, Fradet and Cote (2013) [6], and Mauldin et al. (2013)[7]). Because there are a limited number of proteins known to interact with Elmo, we performed a yeast two-hybrid screen using Elmo1 as bait to identify Elmo1-interacting proteins and to evaluate their mode of regulation. Arhgef16 was one of the proteins identified through the screen and subsequent analyses revealed that Arhgef16 interacted with Elmo1 in mammalian cells as well. Expression of Arhgef16 in phagocytes promoted engulfment of apoptotic cells, and engulfment mediated by Arhgef16 increased synergistically in the presence of Elmo1 but was abrogated in the absence of Elmo1. In addition, Arhgef16-mediated removal of apoptotic cells was dependent on RhoG, but independent of Dock1. Taken together, this study suggests that the newly identified Elmo1-interacting protein, Arhgef16, functions synergistically with Elmo1 to promote clearance of apoptotic cells in a RhoG-dependent and Dock1-independent manner.     

Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180.ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO.Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.     

ClipR-59 Interacts with Elmo2 and Modulates Myoblast Fusion

Recent studies using ClipR-59 knock-out mice implicated this protein in the regulation of muscle function. In this report, we have examined the role of ClipR-59 in muscle differentiation and found that ClipR-59 knockdown in C2C12 cells suppressed myoblast fusion. To elucidate the molecular mechanism whereby ClipR-59 regulates myoblast fusion, we carried out a yeast two-hybrid screen using ClipR-59 as the bait and identified Elmo2, a member of the Engulfment and cell motility protein family, as a novel ClipR-59-associated protein. We showed that the interaction between ClipR-59 and Elmo2 was mediated by the atypical PH domain of Elmo2 and the Glu-Pro-rich domain of ClipR-59 and regulated by Rho-GTPase. We have examined the impact of ClipR-59 on Elmo2 downstream signaling and found that interaction of ClipR-59 with Elmo2 enhanced Rac1 activation. Collectively, our studies demonstrate that formation of an Elmo2·ClipR-59 complex plays an important role in myoblast fusion.     

Control of Vascular Permeability by Atrial Natriuretic Peptide via a GEF-H1-dependent Mechanism

Microtubule (MT) dynamics is involved in a variety of cell functions, including control of the endothelial cell (EC) barrier. Release of Rho-specific nucleotide exchange factor GEF-H1 from microtubules activates the Rho pathway of EC permeability. In turn, pathologic vascular leak can be prevented by treatment with atrial natriuretic peptide (ANP). This study investigated a novel mechanism of vascular barrier protection by ANP via modulation of GEF-H1 function. In pulmonary ECs, ANP suppressed thrombin-induced disassembly of peripheral MT and attenuated Rho signaling and cell retraction. ANP effects were mediated by the Rac1 GTPase effector PAK1. Activation of Rac1-PAK1 promoted PAK1 interaction with the Rho activator GEF-H1, inducing phosphorylation of total and MT-bound GEF-H1 and leading to attenuation of Rho-dependent actin remodeling. In vivo, ANP attenuated lung injury caused by excessive mechanical ventilation and TRAP peptide (TRAP/HTV), which was further exacerbated in ANP^−/− mice. The protective effects of ANP against TRAP/HTV-induced lung injury were linked to the increased pool of stabilized MT and inactivation of Rho signaling via ANP-induced, PAK1-dependent inhibitory phosphorylation of GEF-H1. This study demonstrates a novel protective mechanism of ANP against pathologic hyperpermeability and suggests a novel pharmacological intervention for the prevention of increased vascular leak via PAK1-dependent modulation of GEF-H1 activity.     

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.     

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.     

A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.     

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion.     

GRASP and IPCEF Promote ARF-to-Rac Signaling and Cell Migration by Coordinating the Association of ARNO/cytohesin 2 with Dock180

ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.     

miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.     

Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.