Filopodia Formation Research Articles

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Retrograde Ret signaling controls sensory pioneer axon outgrowth.

The trafficking mechanisms and transcriptional targets downstream of long-range neurotrophic factor ligand/receptor signaling that promote axon growth are incompletely understood. Zebrafish carrying a null mutation in a neurotrophic factor receptor, Ret, displayed defects in peripheral sensory axon growth cone morphology and dynamics. Ret receptor was highly enriched in sensory pioneer neurons and Ret51 isoform was required for pioneer axon outgrowth. Loss-of-function of a cargo adaptor, Jip3, partially phenocopied Ret axonal defects, led to accumulation of activated Ret in pioneer growth cones, and reduced retrograde Ret51 transport. Jip3 and Ret51 were also retrogradely co-transported, ultimately suggesting Jip3 is a retrograde adapter of active Ret51. Finally, loss of Ret reduced transcription and growth cone localization of Myosin-X, an initiator of filopodial formation. These results show a specific role for Ret51 in pioneer axon growth, and suggest a critical role for long-range retrograde Ret signaling in regulating growth cone dynamics through downstream transcriptional changes.

Platelet spreading on fibrinogen matrix, a reliable and sensitive marker of platelet functional activity during storage.

Upon platelet activation, inside-out signals synergistically induced by a variety of agonists and adhesion molecules can enhance the affinity of platelet main integrin, αIIbβ3 to its ligands. Integrin ligation with fibrinogen induces potent signals which develop platelet function including aggregation, release and spreading of which platelet spreading is considered as a major early consequence of αIIbβ3 outside-in signaling. Study presented here evaluated platelet spreading on fibrinogen matrix as a marker of platelet activation during storage. PRP-platelet concentrates were subjected to flowcytometry analysis and the expression levels of P-selectin, CD61, GPIbα and active conformation of the αIIbβ3 (PAC-1 binding) were examined on day 0, 1, 3 and 5 post-storage. Concurrently platelet adhesion and spreading on fibrinogen matrix, glucose concentration and LDH activity were also determined at the same intervals. Results showed significant decreases in platelet spreading on fibrinogen matrix during storage. Spreading was dominant pattern of adhesion till the first day of storage. In 3day-stored platelets, filopodial or lamellipodial formation was dominant event whereas 5day-stored platelets simply adhered to fibrinogen with less protrusion formation and partially failed to spread. Compared to simple adhesion, reduction of platelet spreading was also more significantly correlated with the usual markers of platelet storage lesion including P-selectin (r = - 0.88; p < 0.0001) and GPIbα expression (r = 0.76; p = 0.0001), PAC-1 binding (r = 0.66; p = 0.001), glucose concentration and LDH activity. Taken together, we introduced platelet spreading on fibrinogen matrix as a reliable and sensitive marker of platelets functional activity during storage. As a valid marker which is directly and obviously relevant to the platelet functional capacities, the rapid reduction of platelet spreading during storage overshadows other markers of platelet storage lesion while raising serious question about the quality of 5day-stored platelets.

Physicochemical and biological investigation of oxygen plasma modified electrospun polyurethane scaffolds for connective tissue engineering application

Abstract In this study, electrospinning was selected to fabricate randomly oriented polyurethane (PU) nanofibers for tissue engineering application, and the surface of scaffolds was exposed to oxygen plasma flow. The morphology structure of the PU scaffolds before and after oxygen plasma treatment was observed using scanning electron microscopy (SEM) micrographs, and the fiber diameter distribution was measured using Image J software. The results demonstrated that oxygen plasma modification reduces the fiber diameter without any other special effects on fiber microstructure. Water drop contact angle and swelling ratio of PU constructs were performed to estimate the water-scaffolds interactions. The results revealed improvement of hydrophilicity by oxygen plasma treatment. Atomic force microscopy test was done to analyze a topological characteristic of the scaffolds, and it was found out that oxygen plasma treatment decreases the roughness of the scaffolds. The biological behavior of the scaffolds was investigated by SEM observation and MTT assay after L-929 fibroblast cells culture. In vitro assays demonstrated biocompatibility, cellular attachments, and filopodia formation on plasma modified samples. These results suggest that oxygen plasma treatment improves the physicochemical and biological properties of PU scaffolds to create a more hydrophilic surface which facilitates cell attachments and proliferation.

The Laminin-α1 Chain-Derived Peptide, AG73, Binds to Syndecans on MDA-231 Breast Cancer Cells and Alters Filopodium Formation.

Breast cancer is one of the most common forms of cancer affecting women in the United States, second only to skin cancers. Although treatments have been developed to combat primary breast cancer, metastasis remains a leading cause of death. An early step of metastasis is cancer cell invasion through the basement membrane. However, this process is not yet well understood. AG73, a synthetic laminin-α1 chain peptide, plays an important role in cell adhesion and has previously been linked to migration, invasion, and metastasis. Thus, we aimed to identify the binding partner of AG73 on breast cancer cells that could mediate cancer progression. We performed adhesion assays using MCF10A, T47D, SUM1315, and MDA-231 breast cell lines and found that AG73 binds to syndecans (Sdcs) 1, 2, and 4. This interaction was inhibited when we silenced Sdcs 1 and/or 4 in MDA-231 cells, indicating the importance of these receptors in this relationship. Through actin staining, we found that silencing of Sdc 1, 2, and 4 expression in MDA-231 cells exhibits a decrease in the length and number of filopodia bound to AG73. Expression of mouse Sdcs 1, 2, and 4 in MDA-231 cells provides rescue in filopodia, and overexpression of Sdcs 1 and 2 leads to increased filopodium length and number. Our findings demonstrate an intrinsic interaction between AG73 in the tumor environment and the Sdcs on breast cancer cells in supporting tumor cell adhesion and invasion through filopodia, an important step in cancer metastasis.

Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

SummaryCytoskeletal cross talk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e., during filopodia formation or cilia organization. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle, thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with spindle microtubules forming during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus centrosomal Arp2/3 complex activity plays a role in the maintenance of genomic integrity during mitosis.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Induction of the Epithelial-to-Mesenchymal Transition of Human Colorectal Cancer by Human TNF-β (Lymphotoxin) and its Reversal by Resveratrol.

Objective: Tumor necrosis factor-beta (TNF-β), as an inflammatory mediator that has been shown to promote tumorigenesis, induces NF-κB. Natural multi-targeted agent resveratrol in turn shows anti-inflammatory and anti-cancer properties. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to turn into a motile state with invasive capacities and is associated with metastasis and development of cancer stem cells (CSC). However, TNF-β-induced EMT and the anti-invasion mechanism of resveratrol on CRC are not yet completely understood. Methods: We investigated the underlying molecular mechanisms of resveratrol on TNF-β/TNF-βR-induced EMT and migration of CRC cells (HCT116, RKO, SW480) in monolayer or 3D alginate cultures. Results: TNF-β, similar to TNF-α, induced significant cell proliferation, morphological change, from an epithelial to a spindle-like mesenchymal shape with the formation of filopodia and lamellipodia associated with the expression of EMT parameters (elevated vimentin and slug, reduced E-cadherin), increased migration/invasion, and formation of CSC in all CRC cells. Interestingly, these effects were dramatically decreased in the presence of resveratrol or anti-TNF-βR with TNF-β co-treatment, inducing biochemical changes to the mesenchymal-epithelial transition (MET), with a planar cell surface and suppressed formation of CSC cells. This was associated with a significant increase in apoptosis. Furthermore, we found that resveratrol suppressed TNF-β-induced NF-κB and NF-κB-regulated gene biomarkers associated with growth, proliferation, and invasion. Finally, TNF-βR interacts directly with focal adhesion kinase (FAK) and NF-κB. Conclusion: These results suggest that resveratrol down-regulates TNF-β/TNF-βR-induced EMT, at least in part via specific suppression of NF-κΒ and FAK in CRC cells.

Maternal exposure to cadmium impairs cognitive development of male offspring by targeting the Coronin-1a signaling pathway

Direct exposure to cadmium (Cd) may induce persistent impairment in learning and memory. However, the outcomes of maternal exposure on the neurological development of offspring are much less clear, and the underlying mechanism leading to toxicity remains undisclosed. Following chronic exposure of female rats during gestation and lactation, low level of Cd was detectable in the cerebral cortex but not in the hippocampus of F1 male offspring. The synapses and neurites in hippocampus were destroyed by high Cd exposure level as evidenced by abnormal morphology and cognitive behavior deficit lasting from childhood to adulthood. The membrane glycoprotein M6a (GPM6A) regulates the filopodium formation, neurite outgrowth and synaptogenesis, and is a possible target which Cd acts upon. The signaling pathway Coronin-1a (CORO1A), Ras-related C3 botulinum toxin substrate 1 (RAC1) and p21-activated kinase 1 (PAK1) promotes GPM6A-induced filopodium formation. Our results showed that maternal exposure dramatically down-regulated the level of CORO1A as well as the expression of downstream effectors RAC1, PAK1 and GPM6A. CORO1A-knockdown by siRNA caused decreases in the expression of RAC1, PAK1 and GPM6A; and siRNA targeting combined with Cd insult further decreased the expression of these proteins. Following CORO1A overexpression, the neurites were lengthened with increased expression of all the effector proteins in SH-SY5Y cells exposed to Cd, confirming the significance of CORO1A in mediating the Cd neurotoxicity. These findings may help to disclose how Cd impairs the learning and cognitive development in children, and facilitate finding of potential therapeutic targets for the treatment of Cd poisoning.

Chrysotobibenzyl inhibition of lung cancer cell migration through Caveolin-1-dependent mediation of the integrin switch and the sensitization of lung cancer cells to cisplatin-mediated apoptosis

BackgroundA Lung cancer death account for approximately 1 in 5 of all cancer-related deaths and is particularly virulent due to its enhanced metastasis and resistance to chemotherapy. Chrysotobibenzyl has been reported to decrease cell metastasis, according to the results of an anchorage-independent growth assay; however, its underlying mechanism has not been investigated yet. PurposeThe aim of this study was to investigate the effect of chrysotobibenzyl on lung cancer cell migration and drug sensitization and its mechanism. MethodsCell viability, cell proliferation and drug sensitization were determined by MTT assay. Cell migration was analyzed using a wound-healing assay. Transwell migration and invasion were analyzed using Boyden chamber assay. Mechanisms of chrysotobibenzyl against metastasis including cell migration, invasion, and epithelial to mesenchymal transition (EMT) were evaluated by Western blot analysis and immunofluorescence. ResultsTreatment with chrysotobibenzyl was applied at concentrations of 0–50 µM and the results showed non-cytotoxicity in human lung cancer cells (H460, H292, A549, and H23) and other non-cancerous human cells (HCT116, primary DP1 and primary DP2). However, 50 µM of chrysotobibenzyl significantly altered cell proliferation in H292 cells at 48 h. In addition, 1–50 µM of chrysotobibenzyl significantly inhibited H460 and H292 cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 48 h, which were correlated with reduced protein levels of integrins β1, β3, and αν, p-FAK, p-AKT, Cdc42, and Cav-1. We also established shRNA-Cav-1-transfected (shCav-1) H460 and H292 cells. shCav-1 transfected cells can decrease cell migration and downregulate the expression of integrins β1, β3, and αν when compared with the control. Moreover, chrysotobibenzyl was shown to suppress EMT indicated by the reduction of EMT markers (Vimentin, Snail, and Slug), and sensitize lung cancer cells to cisplatin-mediated apoptosis. ConclusionTreatment with chrysotobibenzyl inhibited lung cancer cell migration via Cav-1, integrins β1, β3, and αν, and EMT suppressions. The downregulation of integrins in response to the compound not only inhibited cell metastasis, but also sensitized lung cancer cells to cisplatin-mediated apoptosis.

Myosin-X Silencing in the Trabecular Meshwork Suggests a Role for Tunneling Nanotubes in Outflow Regulation.

PurposeThe actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM.MethodsShort hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed.ResultsCK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake.ConclusionsInhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.

Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

Angiogenesis is a crucial defense response to hypoxia that regulates the process of raising the promise of long-term neurologic recovery during the management of stroke. A high expression of antiangiogenic factors leads to the loss of neovascularization capacity in pathologic conditions. We have previously documented an impairment of the cerebral vessel perfusion and neovascularization in the cortex neighboring the stroke-induced lesion, which was accompanied by an activation of semaphorin 3E (Sema3E)/PlexinD1 after ischemic stroke. In this study, we employed micro-optical sectioning tomography to fully investigate the details of the vascular pattern, including the capillaries. We found that after transient middle cerebral artery occlusion, inhibiting PlexinD1 signaling led to an organized recovery of the vascular network in the ischemic area. We then further explored the possible mechanisms. In vivo, Sema3E substantially decreased dynamic delta-like 4 (DLL4) expression. In cultured brain microvascular endothelial cells, Sema3E down-regulated DLL4 expression via inhibiting Ras-related C3 botulinum toxin substrate 1-induced JNK phosphorylation. At the microcosmic level, Sema3E/PlexinD1 signaling promoted F-actin disassembly and focal adhesion reduction by activating the small guanosine triphosphatase Ras homolog family member J by releasing RhoGEF Tuba from direct binding to PlexinD1, thus mediating endothelial cell motility and filopodia retraction. Our study reveals that Sema3E/PlexinD1 signaling, which suppressed endothelial DLL4 expression, cell motility, and filopodia formation, is expected to be a novel druggable target for angiogenesis during poststroke progression.-Zhou, Y.-F., Chen, A.-Q., Wu, J.-H., Mao, L., Xia, Y.-P., Jin, H.-J., He, Q.-W., Miao, Q. R., Yue, Z.-Y., Liu, X.-L., Huang, M., Li, Y.-N., Hu, B. Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

GPR124 facilitates pericyte polarization and migration by regulating the formation of filopodia during ischemic injury.

Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive.Methods: A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains.Results: GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration.Conclusion: Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.

A spatially varying charge model for regulating site-selective protein adsorption and cell behaviors.

Implanted materials that enter the body first interact with proteins in body fluids, and cells then perceive and respond to the foreign implant through this layer of adsorbed proteins. Thus, spatially specific regulation of protein adsorption on an implant surface is pivotal for mediating subsequent cellular behaviors. Unlike the surface modulation strategy for traditional biomaterials, in this research, materials with a nonuniform spatial distribution of surface charges were designed to achieve site-selective protein adsorption and further influence cell behavior by charge regulation. Spatially varying microdomains with different levels of piezoelectricity were generated via a focus laser beam-induced phase transition. In addition, after polarization, the zones with different levels of piezoelectricity showed significant differences in surface charge density. The results of scanning Kelvin probe force microscopy (SKPM) showed that the surface charge on the material exhibits a nonuniform spatial distribution after laser irradiation and polarization. Site-specific charge-mediated selective protein adsorption was demonstrated through a protein adsorption experiment. Cell behavior analysis showed that the increase in charge density was conducive to promoting cell adhesion and the formation of filopodia while the nonuniform spatial distribution of charge promoted an oriented arrangement of cells; both features accelerated cell migration. This study provides a new method for spatially regulating protein adsorption through surface charges to further influence cell behaviors.

Centrosomal Actin Assembly is Required for Proper Mitotic Spindle Formation and Chromosome Congression

Cytoskeletal crosstalk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e. during filopodia formation or cilia organisation. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with forming spindle microtubules during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin formation. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus, centrosomal Arp2/3 complex activity plays a role for maintenance of genomic integrity during mitosis.

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phosphorylation of cortactin by cyclin-dependent kinase5 modulates actin bundling by the dynamin 1-cortactin ring-like complex and formation of filopodia and lamellipodia in NG108-15 glioma-derived cells.

Dynamin copolymerizes with cortactin to form a ring-like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin-cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin-dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin-cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F-actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring-like complex with dynamin 1. Consistently, the dynamin 1-phosphomimetic cortactin complexes exhibited decreased actin-bundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG108-15 glioma-derived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactin-dependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA–mDia2 signaling and filopodium formation

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin–binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA–mDia2–regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

Reduced Expression of DOCK4 Leads to Increased Protein Phosphorylation and Migration of Hematopoietic Stem/Progenitor Cells

Dedicator of cytokinesis 4 (DOCK4) is a large, multi-domain signaling protein involved in modulating signaling networks in mammalian cells. Recent studies have highlighted its role across multiple tissue types in maintaining homeostasis. Deletion and/or mutations in the DOCK4 gene lead to malignant conditions and has been attributed to poor long-term prognosis. Within the hematopoietic compartment reduced expression of DOCK4 leads to dysplasia in the erythroid lineage (Sundaravel et al, PNAS, 2015). However, its functions during early stages of hematopoietic cell development have not been defined. Identification of signaling networks regulated by DOCK4 within the hematopoietic compartment will provide insights into developing strategies in overcoming functional deficits that arise due to reduced expression of DOCK4. In this study, using primary human hematopoietic stem/early progenitors (HSPCs), we identified the major downstream signaling networks regulated by DOCK4 and functional consequences of reduced expression of DOCK4.Global phospho-proteomic analysis was performed on primary human CD34+/CD90+ hematopoietic cells expressing normal levels or reduced levels of DOCK4. A similar set of experiments were also performed on cells after they have committed to the erythroid lineage. These initial studies, using 1D and 2D phospho immunoblot analysis, we showed that cells expressing reduced levels of DOCK4 exhibit increased global tyrosine phosphorylation compared to cells expressing normal levels of DOCK4 regardless of whether cells exposed to hematopoietic cytokines. Further analysis of CD34+/CD90+ cells using liquid chromatography coupled mass spectrometry (LC/MS/MS) enabled us to precisely identify multiple proteins that were differentially phosphorylated. Among the proteins that were hyper-phosphorylated, phosphatases, PTPN6 (SHP1), INPP5D (SHIP1) and LYN kinase exhibited the greatest increase in their levels of tyrosine phosphorylation. These results were further validated by immunoblot analysis using site-specific phospho-antibodies against each of the three proteins. Co-immunoprecipitation experiments using HSPCs showed that DOCK4 interacts directly with INPP5D but not with PTPN6 or LYN suggesting that INPP5D is part of the DOCK4 protein complex but not PTPN6 or LYN. We then abrogated DOCK4-INPP5D interaction by knockdown of DOCK4, which led to increased localization of phospho-INPP5D (Y1021) from the cytoplasmic compartment to the cell membrane in CD34+/CD90+ HSPCs. To determine the functional pathways impacted by reduced expression of DOCK4, we performed in-silico DAVID/GO pathway analysis using our phospho proteomic data and found that cell migration function as one of the highly enriched pathways affected, when DOCK4 levels were reduced in HSPCs. We confirmed these findings by examining the migratory abilities of HSPCs expressing low levels of DOCK4 by an in vitro transwell assay, which showed approximately 2-fold increase in HSPC migration in the cell population where DOCK4 was expressed at fifty percent level. In addition, these cells also exhibited increased cell spreading and filopodia formation. Finally, pharmacologic inhibition of enzyme activities/phosphorylation of INPP5D, LYN or PTPN6 decreased the migration of HSPCs. Based on these results; we conclude that DOCK4 is a critical signaling intermediate, which acts within the hematopoietic stem/early progenitor compartment to regulate primary human HSPCs migration by modulating downstream kinases and phosphatases. Therefore using pharmacological inhibitors of INPP5D, LYN or PTPN6 may provide an avenue to restore proper migratory patterns of HSPCs within the hematopoietic niche and potentially reverse differentiation block as observed in dysplasia. DisclosuresNo relevant conflicts of interest to declare.

Single‐Step Approach to Tailor Surface Chemistry and Potential on Electrospun PCL Fibers for Tissue Engineering Application

AbstractA single‐step electrospinning approach enables controlling surface potential of fibers by changing voltage polarities during scaffolds production to enhance cells biointegration. This innovative and facile way of fibers production regulates the interfacial properties to enhance cells adhesion and filopodia formation on fibrous tissue scaffolds for possible bone regeneration. Tuning surface chemistry of polycaprolactone (PCL) by altering voltage polarity during electrospinning allows to double the surface potential on fibers up to 145 mV, which is directly measured using Kelvin probe force microscopy. The obtained surface potential on PCL fibers is directly correlated with surface chemistry analyzed at the grazing angle by X‐ray photoelectron spectroscopy, showing lower oxygen content at PCL fiber surfaces, produced with negative voltage polarity, PCL (−). These fibers create well‐engineered scaffolds that are able to increase significantly cell proliferation that is visualized with fluorescence microscopy, and filopodia formation on positively charged fibers, investigated with high‐resolution scanning electron microscopy. This work introduces electrospun PCL fibers without a need for chemical modification to tune electrostatic interactions between cells and fibrous scaffolds for biomaterials used in regenerative medicine.