Actin-binding Protein Cortactin Research Articles

Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Respiratory syncytial virus disrupts the airway epithelial barrier by decreasing cortactin and destabilizing F-actin.

ABSTRACTRespiratory syncytial virus (RSV) infection is the leading cause of acute lower respiratory tract infection in young children worldwide. Our group recently revealed that RSV infection disrupts the airway epithelial barrier in vitro and in vivo. However, the underlying molecular pathways were still elusive. Here, we report the critical roles of the filamentous actin (F-actin) network and actin-binding protein cortactin in RSV infection. We found that RSV infection causes F-actin depolymerization in 16HBE cells, and that stabilizing the F-actin network in infected cells reverses the epithelial barrier disruption. RSV infection also leads to significantly decreased cortactin in vitro and in vivo. Cortactin-knockout 16HBE cells presented barrier dysfunction, whereas overexpression of cortactin protected the epithelial barrier against RSV. The activity of Rap1 (which has Rap1A and Rap1B forms), one downstream target of cortactin, declined after RSV infection as well as in cortactin-knockout cells. Moreover, activating Rap1 attenuated RSV-induced epithelial barrier disruption. Our study proposes a key mechanism in which RSV disrupts the airway epithelial barrier via attenuating cortactin expression and destabilizing the F-actin network. The identified pathways will provide new targets for therapeutic intervention toward RSV-related disease. This article has an associated First Person interview with the first author of the paper.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Importance of cortactin for efficient epithelial NF-ĸB activation by Helicobacter pylori, Salmonella enterica and Pseudomonas aeruginosa, but not Campylobacter spp.

Transcription factors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family control important signaling pathways in the regulation of the host innate immune system. Various bacterial pathogens in the human gastrointestinal tract induce NF-κB activity and provoke proinflammatory signaling events in infected epithelial cells. NF-κB activation requires the phosphorylation-dependent proteolysis of inhibitor of κB (IκB) molecules including the NF-κB precursors through ubiquitin-mediated proteolysis. The canonical NF-κB pathway merges on IκB kinases (IKKs), which are required for signal transduction. Using CRISPR-Cas9 technology, secreted embryonic alkaline phosphatase (SEAP) reporter assays and cytokine enzyme-linked immunosorbent assay (ELISA), we demonstrate that the actin-binding protein cortactin is involved in NF-κB activation and subsequent interleukin-8 (IL-8) production upon infection by Helicobacter pylori, Salmonella enterica and Pseudomonas aeruginosa. Our data indicate that cortactin is needed to efficiently activate the c-Sarcoma (Src) kinase, which can positively stimulate NF-κB during infection. In contrast, cortactin is not involved in activation of NF-κB and IL-8 expression upon infection with Campylobacter species C. jejuni, C. coli or C. consisus, suggesting that Campylobacter species pluralis (spp.) induce a different signaling pathway upstream of cortactin to trigger the innate immune response.

Helicobacter pylori CagA Induces Cortactin Y-470 Phosphorylation-Dependent Gastric Epithelial Cell Scattering via Abl, Vav2 and Rac1 Activation.

Simple SummaryVarious microbial pathogens target the actin-binding protein cortactin to promote their own uptake, proliferation and spread, and exhibit proposed roles in human cancerogenesis. We aimed to study the molecular mechanisms of how the gastric pathogen Helicobacter pylori hijacks cortactin phosphorylation via tyrosine kinase Abl to trigger cancer-related signal transduction events. We discovered that cortactin phosphorylated at Y-470 recruits the signaling factor Vav2 to activate the small Rho GTPase Rac1, and finally, a cancer cell motility phenotype. We also demonstrate that phosphorylation of cortactin at Y-470 can be completely inhibited by the well-known Abl inhibitor imatinib. Imatinib is an established oral chemotherapy medication, employed for efficient systemic treatment of various cancers. These results reveal a comprehensive novel pathway for how precisely H. pylori manipulates host signaling in gastric disease development, and may pave the way for new opportunities of treatment of the outcome of infections with this pathogen, i.e., through using imatinib.The pathogen Helicobacter pylori is the first reported bacterial type-1 carcinogen playing a role in the development of human malignancies, including gastric adenocarcinoma. Cancer cell motility is an important process in this scenario, however, the molecular mechanisms are still not fully understood. Here, we demonstrate that H. pylori subverts the actin-binding protein cortactin through its type-IV secretion system and injected oncoprotein CagA, e.g., by inducing tyrosine phosphorylation of cortactin at Y-470, which triggers gastric epithelial cell scattering and motility. During infection of AGS cells, cortactin was discovered to undergo tyrosine dephosphorylation at residues Y-421 and Y-486, which is mediated through inactivation of Src kinase. However, H. pylori also profoundly activates tyrosine kinase Abl, which simultaneously phosphorylates cortactin at Y-470. Phosphorylated cortactin interacts with the SH2-domain of Vav2, a guanine nucleotide exchange factor for the Rho-family of GTPases. The cortactin/Vav2 complex then stimulates a previously unrecognized activation cascade including the small GTPase Rac1, to effect actin rearrangements and cell scattering. We hypothesize that injected CagA targets cortactin to locally open the gastric epithelium in order to get access to certain nutrients. This may disturb the cellular barrier functions, likely contributing to the induction of cell motility, which is important in gastric cancer development.

Cortactin Contributes to Activity-Dependent Modulation of Spine Actin Dynamics and Spatial Memory Formation.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

Inhibition of invadopodia formation by diosgenin in tumor cells.

Diosgenin is a type of steroid extracted from the rhizome of Dioscorea plants. In traditional Chinese medicine, Dioscorea has the effect of ‘eliminating phlegm, promoting digestion, relaxing tendons, promoting blood circulation and inhibiting malaria’. Recent studies have confirmed that diosgenin exhibits a number of pharmacological effects, including antitumor activities. Through its antitumor effect, diosgenin is able to block tumor progression and increase the survival rate of patients with cancer; ultimately improving their quality of life. However, the mechanism underlying its pharmacological action remains unclear. Once tumor cells reach a metastatic phase, it can be fatal. Increased migration and invasiveness are the hallmarks of metastatic tumor cells. Invadopodia formation is key to maintaining the high migration and invasive ability of tumor cells. Invadopodia are a type of membrane structure process rich in filamentous-actin and are common in highly invasive tumor cells. In addition to actin, numerous actin regulators, including cortical actin-binding protein (Cortactin), accumulate in invadopodia. Cortactin is a microfilament actin-binding protein with special repetitive domains that are directly involved in the formation of the cortical microfilament actin cell skeleton. Cortactin is also one of the main substrates of intracellular Src-type tyrosine protein kinases and represents a highly conserved family of intracellular cortical signaling proteins. In recent years, great progress has been made in understanding the role of Cortactin and its molecular mechanism in cell motility. However, the diosgenin-Cortactin-invadopodia mechanism is still under investigation. Therefore, the present review focused on the current research on the regulation of invadopodia by diosgenin via Cortactin.

Neural defects caused by total and Wnt1-Cre mediated ablation of p120ctn in mice

Backgroundp120 catenin (p120ctn) is an important component in the cadherin-catenin cell adhesion complex because it stabilizes cadherin-mediated intercellular junctions. Outside these junctions, p120ctn is actively involved in the regulation of small GTPases of the Rho family, in actomyosin dynamics and in transcription regulation. We and others reported that loss of p120ctn in mouse embryos results in an embryonic lethal phenotype, but the exact developmental role of p120ctn during brain formation has not been reported.ResultsWe combined floxed p120ctn mice with Del-Cre or Wnt1-Cre mice to deplete p120ctn from either all cells or specific brain and neural crest cells. Complete loss of p120ctn in mid-gestation embryos resulted in an aberrant morphology, including growth retardation, failure to switch from lordotic to fetal posture, and defective neural tube formation and neurogenesis. By expressing a wild-type p120ctn from the ROSA26 locus in p120ctn-null mouse embryonic stem cells, we could partially rescue neurogenesis. To further investigate the developmental role of p120ctn in neural tube formation, we generated conditional p120ctnfl/fl;Wnt1Cre knockout mice. p120ctn deletion in Wnt1-expressing cells resulted in neural tube closure defects (NTDs) and craniofacial abnormalities. These defects could not be correlated with misregulation of brain marker genes or cell proliferation. In contrast, we found that p120ctn is required for proper expression of the cell adhesion components N-cadherin, E-cadherin and β-catenin, and of actin-binding proteins cortactin and Shroom3 at the apical side of neural folds. This region is of critical importance for closure of neural folds. Surprisingly, the lateral side of mutant neural folds showed loss of p120ctn, but not of N-cadherin, β-catenin or cortactin.ConclusionsThese results indicate that p120ctn is required for neurogenesis and neurulation. Elimination of p120ctn in cells expressing Wnt1 affects neural tube closure by hampering correct formation of specific adhesion and actomyosin complexes at the apical side of neural folds. Collectively, our results demonstrate the crucial role of p120ctn during brain morphogenesis.

SHP2-Independent Tyrosine Dephosphorylation of Cortactin and Vinculin during Infection with Helicobacter pylori.

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

Actin-Binding Protein Cortactin Promotes Pathogenesis of Experimental Autoimmune Encephalomyelitis by Supporting Leukocyte Infiltration into the Central Nervous System.

Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance but is also the basis for the development of pathologic inflammatory conditions within the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we use cortactin gene-inactivated male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin gene-deficient female mice compared with WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and WT splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination, and reduced expression levels of proinflammatory cytokines within the neural tissue compared with WT littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENT Multiple sclerosis is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the CNS where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in experimental autoimmune encephalomyelitis. Leukocyte infiltration into the CNS was inhibited in cortactin-deficient mice, and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of multiple sclerosis.

Bmal1-deficiency affects glial synaptic coverage of the hippocampal mossy fiber synapse and the actin cytoskeleton in astrocytes.

Bmal1 is an essential component of the molecular clockwork, which drives circadian rhythms in cell function. In Bmal1-deficient (Bmal1-/-) mice, chronodisruption is associated with cognitive deficits and progressive brain pathology including astrocytosis indicated by increased expression of glial fibrillary acidic protein (GFAP). However, relatively little is known about the impact of Bmal1-deficiency on astrocyte morphology prior to astrocytosis. Therefore, in this study we analysed astrocyte morphology in young (6-8 weeks old) adult Bmal1-/- mice. At this age, overall GFAP immunoreactivity was not increased in Bmal1-deficient mice. At the ultrastructural level, we found a decrease in the volume fraction of the fine astrocytic processes that cover the hippocampal mossy fiber synapse, suggesting an impairment of perisynaptic processes and their contribution to neurotransmission. For further analyses of actin cytoskeleton, which is essential for distal process formation, we used cultured Bmal1-/- astrocytes. Bmal1-/- astrocytes showed an impaired formation of actin stress fibers. Moreover, Bmal1-/- astrocytes showed reduced levels of the actin-binding protein cortactin (CTTN). Cttn promoter region contains an E-Box like element and chromatin immunoprecipitation revealed that Cttn is a potential Bmal1 target gene. In addition, the level of GTP-bound (active) Rho-GTPase (Rho-GTP) was reduced in Bmal1-/- astrocytes. In summary, our data demonstrate that Bmal1-deficiency affects morphology of the fine astrocyte processes prior to strong upregulation of GFAP, presumably because of impaired Cttn expression and reduced Rho-GTP activation. These morphological changes might result in altered synaptic function and, thereby, relate to cognitive deficits in chronodisruption.

Cortactin Phosphorylation by Casein Kinase 2 Regulates Actin-Related Protein 2/3 Complex Activity, Invadopodia Function, and Tumor Cell Invasion.

Malregulation of the actin cytoskeleton enhances tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin-related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of cortactin T24 by CK2α impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. IMPLICATIONS: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.

Effect of phosphorylation of cortactin at different sites on secretion by airway mucus 5AC

To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites. Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin. Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05). Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.

Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function

Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function.

Cortactin stabilization of actin requires actin-binding repeats and linker, is disrupted by specific substitutions, and is independent of nucleotide state

The actin-binding protein cortactin promotes the formation and maintenance of actin-rich structures, including lamellipodial protrusions in fibroblasts and neuronal dendritic spines. Cortactin cellular functions have been attributed to its activation of the Arp2/3 complex, which stimulates actin branch nucleation, and to its recruitment of Rho family GTPase regulators. Cortactin also binds actin filaments and significantly slows filament depolymerization, but the mechanism by which it does so and the relationship between actin binding and stabilization are unclear. Here we elucidated the cortactin regions that are necessary and sufficient for actin filament binding and stabilization. Using actin cosedimentation assays, we found that the cortactin repeat region binds actin but that the adjacent linker region is required for binding with the same affinity as full-length cortactin. Using total internal reflection fluorescence microscopy to measure the rates of single filament actin depolymerization, we observed that cortactin-actin interactions are sufficient to stabilize actin filaments. Moreover, conserved charged residues in repeat 4 were necessary for high-affinity actin binding, and substitution of these residues significantly impaired cortactin-mediated actin stabilization. Cortactin bound actin with higher affinity than did its paralog, hematopoietic cell-specific Lyn substrate 1 (HS1), and the effects on actin stability were specific to cortactin. Finally, cortactin stabilized ADP-actin filaments, indicating that the stabilization mechanism does not depend on the actin nucleotide state. Together, these results indicate that cortactin binding to actin is necessary and sufficient to stabilize filaments in a concentration-dependent manner, specific to conserved residues in the cortactin repeats, and independent of the actin nucleotide state.

Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC)

Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear. In this study, we propose a new component for the protein machinery involved in the regulation of ENaC, the missing-in-metastasis (MIM) protein. The MIM protein contains an IMD domain (for interaction with PIP2 -rich plasma membrane regions and Rac GTPases; this domain also possesses F-actin bundling activity), a PRD domain (for interaction with cortactin), and a WH2 domain (interaction with G-actin). The patch-clamp electrophysiological technique in whole-cell configuration was used to test the involvement of MIM in the actin-dependent regulation of ENaC. Co-transfection of ENaC subunits with the wild-type MIM protein (or its mutant forms) caused a significant reduction in ENaC-mediated integral ion currents. The analysis of the F-actin structure after the transfection of MIM plasmids showed the important role played by the domains PRD and WH2 of the MIM protein in cytoskeletal rearrangements. These results suggest that the MIM protein may be a part of the complex of actin-binding proteins which is responsible for the actin-dependent regulation of ENaC in the CCD.

Cell scientist to watch – Paul Timpson

ABSTRACT Paul Timpson received his bachelor's degree from the University of Strathclyde, UK and completed his PhD under the supervision of Margaret Frame at the Beatson Institute for Cancer Research and the University of Glasgow in 2002, working on Src kinases and Rho family GTPases in cancer cell invasion. He then moved to the Garvan Institute of Medical Research in Sydney, Australia to work with Roger Daly on the actin-binding protein cortactin in several cancer models. Paul returned to the Beatson Institute in 2007 with the aid of an AstraZeneca postdoctoral research fellowship. He worked with Kurt Anderson on the development of multi-disciplinary live-cell imaging techniques to investigate the molecular dynamics of cancer cells in vivo. In 2012, he started his own research group at the Garvan Institute, assessing cancer in the context of its surrounding environment with the use of advanced intravital imaging technology. His aim is to target distinct hallmarks of cancer progression and to reduce resistance pathways to improve anti-cancer therapeutics.

The actin binding proteins cortactin and HS1 are dispensable for platelet actin nodule and megakaryocyte podosome formation

A dynamic, properly organised actin cytoskeleton is critical for the production and haemostatic function of platelets. The Wiskott Aldrich Syndrome protein (WASp) and Actin-Related Proteins 2 & 3 Complex (Arp2/3 complex) are critical mediators of actin polymerisation and organisation in many cell types. In platelets and megakaryocytes, these proteins have been shown to be important for proper platelet production and function. The cortactin family of proteins (Cttn & HS1) are known to regulate WASp-Arp2/3-mediated actin polymerisation in other cell types and so here we address the role of these proteins in platelets using knockout mouse models.We generated mice lacking Cttn and HS1 in the megakaryocyte/platelet lineage. These mice had normal platelet production, with platelet number, size and surface receptor profile comparable to controls. Platelet function was also unaffected by loss of Cttn/HS1 with no differences observed in a range of platelet function assays including aggregation, secretion, spreading, clot retraction or tyrosine phosphorylation. No effect on tail bleeding time or in thrombosis models was observed. In addition, platelet actin nodules, and megakaryocyte podosomes, actin-based structures known to be dependent on WASp and the Arp2/3 complex, formed normally. We conclude that despite the importance of WASp and the Arp2/3 complex in regulating F-actin dynamics in many cells types, the role of cortactin in their regulation appears to be fulfilled by other proteins in platelets.

Abstract 5067: Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function

Abstract Malregulation of signal transduction events controlling cortical actin cytoskeleton dynamics is responsible for to enhancing tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in many cancers, corresponding with increased tumor progression and poor patient outcome. In many cancer types, elevated Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. While cortactin-mediated activation of Arp2/3 complex has been well established, the objective of this study is to identify upstream signaling pathways responsible for modulating the interaction between cortactin and Arp2/3 complex in invasive carcinoma cells. We have determined that casein kinase (CK) 2α is responsible for disrupting the ability of cortactin to bind and/or activate Arp2/3 complex. CK2α directly phosphorylates cortactin at a conserved threonine 24 (T24) adjacent to the canonical Arp2/3 binding motif in the cortactin amino-terminal acidic (NTA) domain. CK2α phosphorylation of cortactin T24 impairs the ability of cortactin to activate Arp2/3 actin nucleation as determined by spectrofluorometric analysis. Decreased invadopodia formation and ECM degradation activity is observed in head and neck squamous cell carcinoma (HNSCC) cells with shRNA-mediated CK2α knockdown, expression of a CK2α phosphorylation-null cortactin mutant (T24A), and with treatment of the Phase I CK2α inhibitor CX-4945 (Silmitasertib). Collectively, these data suggest that CK2α-mediated cortactin phosphorylation at T24 is a critical event in regulating the interaction with Arp2/3 complex, identifying a key anti-invasive mechanism impacted by targeted anti-CK2α therapeutics. Citation Format: Steven M. Markwell, Amanda Gatesman Ammer, Erik T. Interval, Dorothy A. Schafer, River A. Hames, Scott A. Weed. Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5067.

Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility

Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.