Cell Filopodia Formation Research Articles

P10.01 Anti-migration and anti-invasion effects of curcumin via suppression of fascin expression in glioblastoma cells

Abstract BACKGROUND The natural compound Curcumin was known to inhibit migration and invasion of glioblastoma (GBM) cells. Fascin, a kind of actin-binding proteins, is correlated with migration and invasion of GBM cells. The purpose of this study was to investigate anti-migration and anti-invasion effects of Curcumin via suppression of fascin expression in GBM cells. MATERIAL AND METHODS U87 cell line was used as an experimental model of GBM. Fascin was quantified by Western blot analysis. And, the signal transducer and activator of transcription 3 (STAT3), known to play an important role in migration and invasion of tumor cells, were analyzed by sandwich-ELISA. Migration and invasion capacities were assessed by attachment, migration and invasion assays. Cellular morphology was demonstrated by immunofluorescence. RESULTS At various concentrations of curcumin and exposure times, fascin expression decreased. After temporarily exposure to 10μM/L Curcumin during 6 hours as less invasive concentration and time, fascin expression temporarily decreased at 12 hours (18.4%, p=0.024), and since then recovered. And, the change of phosphrylated STAT3 level also reflected the temporarily decreased pattern of fascin expression at 12 hours (19.7%, p=0.010). Attachment, migration, and invasion capacities consistently decreased at 6, 12, and 24 hours. And, immunofluorescence showed the change of shape and the reduction of filopodia formation in cells. CONCLUSION Curcumin is likely to suppress the fascin expression in GBM cells, and this might be a possible mechanism for anti-migration and anti-invasion effects of Curcumin via inhibition of STAT3 phosphorylation.

Selective IKK2 inhibitor IMD0354 disrupts NF-\u03baB signaling to suppress corneal inflammation and angiogenesis

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase β (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.

Actin organization and dynamics: novel regulatory mechanisms from the biophysical to the tissue level

Properly regulated actin organization and dynamics are crucial for cellular processes such as cell migration, cell division, and maintenance of cell–cell junctions. Speakers in the “Actin Organization and Dynamics” Minisymposium at the ASCB 2012 meeting highlighted new and interesting mechanisms that underlie the regulation of actin dynamics. The session was broad in scope, ranging from in vitro biochemical and biophysical studies with purified components to cellular- and tissue-level studies. There was an emphasis on severing mechanisms and mechanics, as well as the complexities of regulatory pathways in cells. Hyeran Kang (Yale University), a postdoc with Enrique M. De La Cruz, presented work on how cations affect filament mechanics and severing via the regulatory protein, cofilin. Kang identified, using structural bioinformatics, two potential filament-specific cation-binding sites and classified them as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent assembly and bending mechanics, respectively. Cofilin enhances actin filament bending and twisting compliance, and it is hypothesized that local mechanical discontinuities in partially decorated filaments promote severing at boundaries of bare and cofilin-decorated segments. Using native and engineered yeast actin mutants, Kang showed that dissociation of stiffness site cations by human cofilin drives changes in filament bending mechanics and that this is required for severing. Pinar Gurel (Geisel School of Medicine at Dartmouth), a graduate student with Harry Higgs, described research investigating the severing activity of INF2, a formin linked to two human genetic diseases: Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis. Using a combination of classic biochemical experiments and total internal reflection fluorescence (TIRF) microscopy, Gurel showed that, in addition to the normal formin binding site at the barbed end, INF2 binds the sides of actin filaments stoichiometrically (one INF2 per subunit) and with submicromolar affinity. INF2-catalyzed filament severing occurs within INF2-decorated regions of partially and fully decorated filaments. This property, along with INF2’s ability to bind ADP-Pi filaments, distinguishes INF2-mediated filament severing from that of cofilin. Dennis Breitsprecher (Brandeis University), a postdoc with Bruce Goode, presented recent work demonstrating a new role for Srv2/cyclase-associated protein. Breitsprecher observed, using dual-color TIRF microscopy, that the N-terminal half of Srv2 (N-Srv2) increases the severing activity of cofilin without appreciably affecting cofilin binding to filaments. Electron microscopy and single-particle analysis revealed that N-Srv2 self-associates into hexameric ring structures. N-terminal truncations weaken N-Srv2 oligomerization, compromise enhanced severing activity, and yield actin organization and cell polarity defects in cells. Peter Bieling (University of California, San Francisco), a postdoc with Dyche Mullins and Dan Fletcher, evaluated how reconstituted dendritic actin network growth responds to external loads. Dendritic actin networks assembled in vitro from micron-sized patterns of surface-immobilized nucleation-promoting factor were challenged with force applied via an atomic force microscope (AFM) cantilever, and actin assembly was monitored using multicolor TIRF microscopy and AFM. Bieling demonstrated that the dendritic actin networks dynamically adapt to external forces; the actin network growth velocity decreases with load, while actin density in the force-generating region of the network increases with load. Barbed-end capping is force-dependent and slows with increasing load. Colleen G. Bilancia (University of North Carolina, Chapel Hill), a postdoc with Mark Peifer, presented work on how Enabled (Ena) and Diaphanous (Dia) coordinate to regulate actin filament barbed-end polymerization. Overexpressing active Dia in Drosophila D16 cells promotes long, stable filopodial protrusions, while overexpressing Ena leads to dynamic, fan-like protrusions. Coexpression of Dia and Ena promotes protrusions distinct from those produced with expression of either regulator independently. Bilancia noted that filopodia retract when active Dia and Ena colocalize, suggesting that Ena negatively regulates Dia, possibly through direct binding interactions. Indeed, Bilancia showed that the Ena EVH1 domain directly binds the Dia FH1 domain, and the EVH1 domain inhibits Dia-mediated in vitro pyrene actin assembly and filopodia formation in cells. Ann Miller (University of Michigan) described her lab's finding that the cytokinesis regulator anillin plays a novel role in regulating epithelial cell–cell junctions. Using Xenopus embryos as a model system, Miller showed that a population of anillin localizes at cell–cell junctions in both dividing and nondividing cells. Further, anillin functionally regulates junctional integrity. Both tight junctions and adherens junctions are disrupted when anillin is knocked down. Anillin binds actin filaments and the small GTPase RhoA, both of which regulate cell–cell junction structure and function. Miller's group tested the effects of anillin expression on RhoA activation and found that anillin knockdown results in increased spontaneous flares of active RhoA that are prominent at cell–cell junctions in both dividing cells and nondividing regions of the epithelium.

Mechanism of Actin Filament Bundling by Fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Expression of neuregulin 4 splice variants in normal human tissues and prostate cancer and their effects on cell motility

The neuregulin 4 gene encodes at least five different variants (designated A1, A2, B1, B2 and B3) produced as a result of alternative splicing. We have determined their sites of expression in normal human adult tissues using isoform-specific antibodies. Their expression is cell type specific and differs in subcellular location suggesting that they may have varied functions in these contexts. We have shown in a panel of prostate cancers that each form is present to differing degrees, and that principal component analysis indicates that there are three patterns of expression. Some isoforms were positively correlated with high prostate-specific antigen levels and others were inversely associated with Gleason score. Synthetic, refolded A forms promoted lamellipodia and filopodia formation in cells expressing the ErbB4 (CTa) receptor and stimulated cell motility in wound healing assays. The data suggest that the different forms have varied sites of expression and function, and this includes effects on cell architecture and motility.