Proteomes, kinases and signalling pathways in virus-induced filopodia, as potential antiviral therapeutics targets.

Filopodia are thin finger-like protrusions at the surface of cells that are internally occupied with bundles of tightly parallel actin filaments. They play significant roles in cellular physiological processes, such as adhesion to extracellular matrix, guidance towards chemo-attractants and in wound healing. Filopodia were recently reported to play important roles in viral infection including initial viral attachment to host cells, cell surfing, viral trafficking, internalization, budding, virus release and spread to other cells in a form that would avoid the host immune system. The detailed virus-host protein interactions underlying most of these processes remain to be elucidated. This review will describe some reported virus-host protein interactions on filopodia with the aim of identifying potential new anti-virus therapeutic targets. Exploring this research area may lead to the development of novel classes of anti-viral therapeutics that can block signalling pathways used by the virus to trigger filopodia formation. Successful compounds would inhibit initial virus attachment, formation of filopodia, expression of putative virus binding protein, extracellular virus trafficking, and budding.

The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis.

TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].

Statin treatment reduces matrix degradation capacity of proinflammatory polarized macrophages.

BACKGROUND AND AIMS: Macrophages are versatile immune cells involved in tissue degradation and remodeling. Proinflammatory macrophages have the highest capacity of matrix degradation and proteolysis. Within atherosclerotic lesions, proinflammatory macrophages are associated with unstable plaques. Statins have been demonstrated to increase plaque stability. Possible changes of polarized macrophage tissue degradation behavior under statin treatment are currently unknown. METHODS: Polarized macrophages were tested in vitro for matrix degradation capacity with or without statin treatment. RESULTS: Proinflammatory macrophages show high matrix degradation capacity, which is lost after statin treatment. Statin concentrations were within a physiological range and did not influence overall macrophage polarization. Proinflammatory macrophages showed however a loss of filopodia where activators of MMPs are located. Loss of matrix degradation in proinflammatory macrophages was associated with changes of MMP14 activation and loss of uPAR localization at filopodia. Supplementation of mevalonate restored localization of uPAR to cellular protrusions and matrix degradation capacity. CONCLUSION: Statins reduce the matrix degradation potential of proinflammatory macrophages by reducing uPAR localization to cellular filopodia and reducing intracellular MMP14 activation.

Activation of the neurokinin 3 receptor promotes filopodia growth and sprouting in rat embryonic hypothalamic cells.

Members of the tachykinin family have trophic effects on developing neurons. The tachykinin neurokinin 3 receptor (NK3R) appears early in embryonic development; during the peak birthdates of hypothalamic neurons, but its involvement in neural development has not been examined. To address its possible role, immortalized embryonic hypothalamic neurons (CLU209) were treated with CellMask, a plasma membrane stain, or the membranes were imaged in CLU209 cells that were transfected with a pEGFP-NK3R expression vector. Nontransfected cells and transfected cells were then treated with senktide, a NK3R agonist, or Dulbecco's Modified Eagle's Medium (DMEM) and time-lapse confocal images were captured for the following 30 min. Compared to DMEM, senktide treatment led to filopodia initiation from the soma of both nontransfected and transfected CLU209 cells. These filopodia had diameters and lengths of approximately 200 nm and 3 µm, respectively. Pretreatment with an IP3 receptor blocker, 2-aminoethoxydiphenyl borate (2-APB), prevented the senktide-induced growth in filopodia; demonstrating that NK3R-induced outgrowth of filopodia likely involves the release of intracellular calcium. Exposure of transfected CLU209 cells to senktide for 24 h led to further growth of filopodia and processes that extended 10-20 µm. A mathematical model, composed of a linear and population model was developed to account for the dynamics of filopodia growth during a timescale of minutes. The results suggest that the ligand-induced activation of NK3R affects early developmental processes by initiating filopodia formation that are a prerequisite for neuritogenesis.

Denbinobin, a phenanthrene from Dendrobium nobile, impairs prostate cancer migration by inhibiting Rac1 activity.

Prostate cancer is the most prevalent type of cancer in the United States. The most common site of prostate cancer metastasis is bone. CXCL12 is preferentially expressed in bone and is targeted by prostate cancer cells, which over-express the receptor for CXCL12, CXCR4. In response to CXCL12 stimulation, Rac1, a GTPase, along with its effectors, regulates actin polymerization to form lamellipodia, which is a critical event for cell migration. Cortactin, an actin-binding protein, is recruited to the lamellipodia and is phosphorylated at tyrosine residues. The phosphorylated cortactin is also involved in cell migration. The inhibition of Rac1 activity using a dominant negative Rac1 impairs lamellipodial protrusion as well as cortactin translocation and cortactin phosphorylation. Denbinobin, a substance extracted from Dendrobium nobile, has anticancer effects in many cancer cell lines. Whether denbinobin can inhibit prostate cancer cell migration is not clear. Here, we report that denbinobin inhibited Rac1 activity. The inhibition of Rac1 activity prevented lamellipodial formation. Cortactin phosphorylation and translocation to the lamellipodia were also impaired, and PC3 cells were unable to migrate. These results indicate that denbinobin prevents CXCL12-induced PC3 cell migration by inhibiting Rac1 activity.

Postprandial triglyceride-rich lipoproteins promote invasion of human coronary artery smooth muscle cells in a fatty-acid manner through PI3k-Rac1-JNK signaling.

SCOPE: The aim was to investigate the effect of postprandial triglyceride-rich lipoproteins (TRLs) with different fatty acid compositions on human coronary artery smooth muscle cell (hCASMC) invasion and to identify the molecular pathways involved. METHODS AND RESULTS: TRLs were isolated from the plasma of healthy volunteers after the ingestion of single meals enriched in MUFAs, saturated fatty acids (SFAs), or PUFAs. hCASMC invasion was analyzed using transwell chambers with Matrigel. TRLs-SFAs provoked the highest invasion, followed by TRLs-MUFAs and TRLs-PUFAs. Inhibition studies with Orlistat showed that invasion was dependent on the fatty acid composition of the TRLs. Fatty acids incorporated into the cell membranes strongly associated with cell invasion. Pull-down assays showed that TRLs-SFAs were able to increase Rac1 activity via inhibition of RhoA-dependent signaling. Chemical inhibition and siRNA studies showed that Rac1, PI3k, JNK, and MMP2 regulates TRL-SFA-induced hCASMC invasion. CONCLUSION: We demonstrate for the first time that TRLs induce hCASMCs invasion in a fatty acid dependent manner. This effect in TRLs-SFAs is mediated by the PI3k-Rac1-JNK, RhoA, and Rac1-MMP2 pathways. The ingestion of MUFA, compared to other dietary fatty acids such as SFA, could be considered as a nutritional strategy to reduce the atherosclerotic plaque formation.

Myosin-X functions in polarized epithelial cells.

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein-Myo10 localizes to lateral membrane cell-cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.

Filopodia and adhesion in cancer cell motility.

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.

Structural basis of the myosin X PH1(N)-PH2-PH1(C) tandem as a specific and acute cellular PI(3,4,5)P(3) sensor.

Myosin X (MyoX) is an unconventional myosin that is known to induce the formation and elongation of filopodia in many cell types. MyoX-induced filopodial induction requires the three PH domains in its tail region, although with unknown underlying molecular mechanisms. MyoX's first PH domain is split into halves by its second PH domain. We show here that the PH1(N)-PH2-PH1(C) tandem allows MyoX to bind to phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P(3)] with high specificity and cooperativity. We further show that PH2 is responsible for the specificity of the PI(3,4,5)P(3) interaction, whereas PH1 functions to enhance the lipid membrane-binding avidity of the tandem. The structure of the MyoX PH1(N)-PH2-PH1(C) tandem reveals that the split PH1, PH2, and the highly conserved interdomain linker sequences together form a rigid supramodule with two lipid-binding pockets positioned side by side for binding to phosphoinositide membrane bilayers with cooperativity. Finally, we demonstrate that disruption of PH2-mediated binding to PI(3,4,5)P(3) abolishes MyoX's function in inducing filopodial formation and elongation.

Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10.

Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca(2+) increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca(2+) is poor, and addition of Ca(2+) decreases the K(d) to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.

Isoalvaxanthone inhibits colon cancer cell proliferation, migration and invasion through inactivating Rac1 and AP-1.

Isoalvaxanthone (IAX) is a bioactive xanthone isolated from Cudrania cochinchinensis (Lour.). However, the function and mechanism of this compound in cancer migration and invasion have not been elucidated to date. In this study, we found that IAX could suppress various steps of tumor metastasis including proliferation, migration and invasion in a dose-dependent manner on colorectal cancer cells. Especially matrix metalloproteinase 2 (MMP-2), the pivotal factor in cancer invasion, was suppressed both on activation and expression after treated with IAX. To understand the underlying mechanism of IAX on the inhibitory effect of proliferation, migration and invasion, we demonstrated that IAX could significantly inhibit the activation of Rac1 but has undetectable effect on GTP-RhoA, GTP-Cdc42 and the phosphorylation of ERK1/2, p38 MAPK and JNK. Moreover, IAX showed little influence on the transcriptional activity of nuclear transcription factor kappaB (NF-kappaB) but strongly inhibited that of activator protein-1 (AP-1), which is the downstream transcriptional factor of Rac1. Together, our results indicate that IAX exerts anticancer effect in SW620 cells by targeting MMP-2 via regulating the activity of Rac1 and AP-1. These results are the first to reveal the function of IAX in tumor metastasis and its underlying molecular mechanism, thus suggest IAX to be a promising antimetastatic agent.

Virtual screening approach for the identification of new Rac1 inhibitors.

Rac1 protein is implicated in several events of atherosclerotic plaque development and represents a new potential pharmacological target for cardiovascular diseases. In this paper we describe a pharmacophore virtual screening followed by molecular docking calculations leading to the identification of five new Rac1 inhibitors. These compounds were shown to be more effective than the reference compound NSC23766 in reducing the intracellular levels of Rac1-GTP, thus supporting this approach for the development of new Rac1 inhibitors.

Interaction with the IQ3 motif of myosin-10 is required for calmodulin-like protein-dependent filopodial extension.

Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.

Phospholipase D is essential for keratocyte-like migration of NBT-II cells.

NBT-II cells on collagen-coated substrates move rapidly and persistently, maintaining a semi-circular shape with a large lamellipodium, in a manner similar to fish keratocytes. The inhibitor of phospholipase D (PLD), n-butanol, completely blocked the migration and disturbed the characteristic localization of actin along the edge of lamellipodia. To investigate the functional difference between the two isozymes of PLD (PLD1 and PLD2), we transfected NBT-II cells with vectors expressing shRNA to deplete PLD1 or PLD2. Depletion of both PLD1 and 2 by RNA interference reduced the velocity of the migration, but depletion of PLD2 inhibited motility more severely than that of PLD1. Furthermore, GFP-PLD2 was localized to the protruding regions of lamellipodia in migrating cells. Thus, PLD is essential for the maintenance of keratocyte-like locomotion of NBT-II cells, presumably by regulating the actin cytoskeleton.

Role of calcium-dependent actin-bundling proteins: characterization of Dictyostelium mutants lacking fimbrin and the 34-kilodalton protein.

Actin-bundling proteins organize actin filaments into densely packed bundles. In Dictyostelium discoideum two abundant proteins display calcium-regulated bundling activity, fimbrin and the 34-kDa protein (ABP34). Using a GFP fusion we observed transient localization of fimbrin at the phagocytic cup and macropinosomes. The distribution of truncated constructs encompassing the EF hands and the first actin-binding domain (EA1) or both actin-binding domains devoid of EF hands (A1A2) was indistinguishable from that of the full length protein. The role of fimbrin and a possible functional overlap with ABP34 was investigated in fim- and double 34-/fim- mutants. Except for a moderate cell size defect, fim- mutants did not show defects in growth, endocytosis, exocytosis, and chemotaxis. Double mutants were characterized by a small cell size and a defect in morphogenesis resulting in small fruiting bodies and a low spore yield. The cell size defect could not be overcome by expression of fimbrin fragments EA1 or A1A2, suggesting that both bundling activity and regulation by calcium are important. Induction of filopod formation in 34-/fim- cells was not impaired, indicating that both proteins are dispensable for this process. We searched in the Dictyostelium genome database for fimbrin-like proteins that could compensate for the fimbrin defect and identified three unconventional fimbrins and two more proteins with actin-binding domains of the type present in fimbrins.

Dental pulp cells provide neurotrophic support for dopaminergic neurons and differentiate into neurons in vitro; implications for tissue engineering and repair in the nervous system.

Glial cell line-derived neurotrophic factor (GDNF) mRNA is highly expressed by dental pulp cells (DPCs) prior to the initiation of dental pulp innervation. We show that radioactively labelled exogenous GDNF is retrogradely transported from neonatal teeth and vibrissae to the trigeminal neurons, indicating that GDNF acts as a classical neurotrophic factor in the trigeminal system. We also show that DPCs from both rats and humans produce nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and GDNF mRNAs in vitro, promote the survival and phenotypic characteristics of embryonic dopaminergic (DA) neurons and protect DA neurons against the neurotoxin 6-hydroxy-dopamine (6-OHDA) in vitro. By using inhibitory antibodies to NGF, BDNF and GDNF, we show that the promotion of DA neuron survival relates to the production and release of neurotrophic proteins by DPCs in vitro. We suggest that in vivo production of neurotrophic factors by DPCs play roles in tooth innervation. However, continued production of neurotrophic factors by the DPCs might have wider implications. We propose that the dental pulp is a viable source of easily attainable cells with possible potential for development of autologous cell transplantation therapies. We also show that a population of neural crest-derived dental pulp cells acquire clear neuronal morphology and protein expression profile in vitro, indicating the presence of a cell population in the dental pulp with neuronal differentiation capacity that might provide additional benefits when grafted into the CNS.

Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules.

We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.

PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia.

The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.