Actin-rich Protrusions Research Articles

Abstract 1108: M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation

Abstract Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer related deaths. Emerging evidences suggest that tumour microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumour results in chronic inflammation which favours tumour growth and progression. Particularly, tumour associated macrophages (TAMs) are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers and is closely associated with EBV infection. We postulate that TAMs may increase the invasiveness of NPC by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were polarized to M2-macrophages, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells then formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the co-culture medium of the M2-macrohphages and the NPC cells. Tumour necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Besides, western blot analysis identified that several actin-associated proteins such as Src, Erk, and cortactin were involved in the TNFα-mediated invadopodia formation. We also found that the expression of latent membrane protein 1 (LMP1), an EBV protein that has similar structure to TNF receptor was elevated in NPC cells after co-culturing with the M2-polarized macrophages. Through transient transfection and stable expression of LMP1 in the NPC cells, LMP1 could also promote the formation of invadopodia through cdc42 activation. Taken together, these data indicate macrophages can promote the invasiveness of EBV-infected NPC cells by enhancing their ability in forming invadopodia through an activated TNFα signaling axis and LMP1 upregulation. Citation Format: Wilson Wing Chung Tang, Chan Ping You, Gareth E. Jones, George Sai-Wah Tsao, Anna Chi Man Tsang. M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1108.

PO-251 High density fibrillar collagen promotes breast cancer cell invasion by decreasing actin dynamics at invadopodia

IntroductionDuring metastasis, cancer cells migrate through the extracellular matrix (ECM) to reach blood and lymphatic vessels and spread into distant sites. Tumour cell invasiveness is associated with the formation of actin-rich protrusions termed invadopodia. These structures promotes ECM degradation and thereby increase cancer cell ability to cross physical barriers. An increased deposition of dense fibrillar collagen has been associated with higher risks of metastasis. In this regard, high-density fibrillar collagen (HDFC), a condition that mimics the dense microenvironment of breast cancer, was reported to be a potent inducer of invadopodia. Invadopodia formation and activity critically rely on the actin cytoskeleton. To get a better insight into the mechanism by which the desmoplastic stroma stimulates invadopodia formation, we explored actin cytoskeleton dynamics in HDFC-induced breast cancer cell invadopodia.Material and methodsFluorescent Recovery After Photobleaching (FRAP) was used to quantify actin dynamics at invadopodia from cancer cells expressing GFP-fused actin and plated on HDFC-coated slides. In addition, the role of two invadopodial actin-bundling proteins associated with metastatic breast cancer (Fascin and CRP2) was evaluated using a RNAi strategy, and their biochemical activities were compared in in vitro reconstituted assays using TIRF microscopy.Results and discussionsOur FRAP analyses revealed that a gelatin matrix induces the formation of invadopodia with identical actin dynamics. In contrast, HDFC induced the formation of an additionnal population of invadopodia with significantly decreased actin dynamics. The proportion of this stable invadopodia population increased over time and was associated with MT1-MMP accumulation. Knocking down actin bundling proteins, such as fascin or CRP2, induced an increase in the mobile actin fraction and a shift toward the dynamic subpopulation. Such effects were associated with a decrease in MT1-MMP levels at invadopodia, and a reduction in the invasive potential of breast cancer cells. TIRF microscopy analyses revealed that only CRP2 protects actin bundles against cofilin-mediated severing, suggesting only partial redundancy between the two actin regulators.ConclusionIn this study we found that HDFC decreases actin dynamics at invadopodia, which in turn increases tumour cell invasive capacity. In addition, our data suggest that such decrease in actin dynamics involves functionally similar, but not identical, actin bundling factors.

Actin-Based Cell Protrusion in a 3D Matrix.

Cell migration controls developmental processes (gastrulation and tissue patterning), tissue homeostasis (wound repair and inflammatory responses), and the pathobiology of diseases (cancer metastasis and inflammation). Understanding how cells move in physiologically relevant environments is of major importance, and the molecular machinery behind cell movement has been well studied on 2D substrates, beginning over half a century ago. Studies over the past decade have begun to reveal the mechanisms that control cell motility within 3D microenvironments – some similar to, and some highly divergent from those found in 2D. In this review we focus on migration and invasion of cells powered by actin, including formation of actin-rich protrusions at the leading edge, and the mechanisms that control nuclear movement in cells moving in a 3D matrix.

Amastigote Synapse: The Tricks of Trypanosoma cruzi Extracellular Amastigotes

To complete its life cycle within the mammalian host, Trypanosoma cruzi, the agent of Chagas’ disease, must enter cells. Trypomastigotes originating from the insect vector (metacyclic) or from infected cells (bloodstream/tissue culture-derived) are the classical infective forms of the parasite and enter mammalian cells in an actin-independent manner. By contrast, amastigotes originating from the premature rupture of infected cells or transformed from swimming trypomastigotes (designated extracellular amastigotes, EAs) require functional intact microfilaments to invade non-phagocytic host cells. Earlier work disclosed the key features of EA-HeLa cell interplay: actin-rich protrusions called ‘cups’ are formed at EA invasion sites on the host cell membrane that are also enriched in actin-binding proteins, integrins and extracellular matrix elements. In the past decades we described the participation of membrane components and secreted factors from EAs as well as the actin-regulating proteins of host cells involved in what we propose to be a phagocytic-like mechanism of parasite uptake. Thus, regarding this new perspective herein we present previously described EA-induced ‘cups’ as parasitic synapse since they can play a role beyond its architecture function. In this review, we focus on recent findings that shed light on the intricate interaction between extracellular amastigotes and non-phagocytic HeLa cells.

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

Septins regulate junctional integrity of endothelial monolayers.

Junctional integrity of endothelial monolayers is crucial to control movement of molecules and cells across the endothelium. Examining the structure and dynamics of cell junctions in endothelial monolayers, we discovered a role for septins. Contacts between adjacent endothelial cells were dynamic, with protrusions extending above or below neighboring cells. Vascular endothelial cadherin (VE-cadherin) was present at cell junctions, with a membrane-associated layer of F-actin. Septins localized at cell-junction membranes, in patterns distinct from VE-cadherin and F-actin. Septins assumed curved and scallop-shaped patterns at junctions, especially in regions of positive membrane curvature associated with actin-rich membrane protrusions. Depletion of septins led to disrupted morphology of VE-cadherin junctions and increased expression of VE-cadherin. In videos, septin-depleted cells displayed remodeling at cell junctions; regions with VE-cadherin were broader, and areas with membrane ruffling were wider. Septin depletion and junction disruption led to functional loss of junctional integrity, revealed by decreased transendothelial electric resistance and increased transmigration of immune cells. We conclude that septins, as cytoskeletal elements associated with the plasma membrane, are important for cell junctions and junctional integrity of endothelial monolayers, functioning at regions of positive curvature in support of actin-rich protrusions to promote cadherin-based cell junctions.

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Our results demonstrate a crucial role for CypA during Listeria infections.

A RAB35-p85/PI3K axis controls oscillatory apical protrusions required for efficient chemotactic migration

How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidence indicates that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of circular dorsal ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as a steering wheel for GF-mediated chemotaxis and chemoinvasion.

The Molecular Targets and Anti-Invasive Effects of 2,6-bis-(4-hydroxyl-3methoxybenzylidine) cyclohexanone or BHMC in MDA-MB-231 Human Breast Cancer Cells.

In order to metastasize, tumor cells need to migrate and invade the surrounding tissues. It is important to identify compound(s) capable of disrupting the metastasis of invasive cancer cells, especially for hindering invadopodia formation, so as to provide anti-metastasis targeted therapy. Invadopodia are thought to be specialized actin-rich protrusions formed by highly invasive cancer cells to degrade the extracellular matrix (ECM). A curcuminoid analogue known as 2,6-bis-(4-hydroxy-3-methoxybenzylidine)cyclohexanone or BHMC has shown good potential in inhibiting inflammation and hyperalgesia. It also possesses an anti-tumor effects on 4T1 murine breast cancer cells in vivo. However, there is still a lack of empirical evidence on how BHMC works in preventing human breast cancer invasion. In this study, we investigated the effect of BHMC on MDA-MB-231 breast cancer cells and its underlying mechanism of action to prevent breast cancer invasion, especially during the formation of invadopodia. All MDA-MB-231 cells, which were exposed to the non-cytotoxic concentrations of BHMC, expressed the proliferating cell nuclear antigen (PCNA), which indicate that the anti-proliferative effects of BHMC did not interfere in the subsequent experiments. By using a scratch migration assay, transwell migration and invasion assays, we determined that BHMC reduces the percentage of migration and invasion of MDA-MB-231 cells. The gelatin degradation assay showed that BHMC reduced the number of cells with invadopodia. Analysis of the proteins involved in the invasion showed that there is a significant reduction in the expressions of Rho guanine nucleotide exchange factor 7 (β-PIX), matrix metalloproteinase-9 (MMP-9), and membrane type 1 matrix metalloproteinase (MT1-MMP) in the presence of BHMC treatment at 12.5 µM. Therefore, it can be postulated that BHMC at 12.5 µM is the optimal concentration for preventing breast cancer invasion.

Collagen morphology influences macrophage shape and marker expression in vitro

Fibrosis is a process in which an accumulation of extracellular matrix (ECM) leads to an impaired function of the affected organ. Pulmonary fibrosis is the end-stage of several lung diseases, characterized by scarring of the lungs. Although macrophages are known to be important players in ECM homeostasis, their ability to respond to fibrosis-related morphological and mechanical changes of the ECM is relatively unexplored. In this study we aimed to elucidate the effect of ECM stiffness and morphology on macrophage polarization, by using a collagen type I-based in vitro system. Collagen morphology, but not stiffness, affected the relative expression of CD206 (the mannose receptor) and Ym1 (a murine marker of pro-healing M2 macrophages). Higher expression of Ym1 was found when macrophages were cultured on fibrous collagen. Globular collagen led to higher expression of CD206, a marker known to be upregulated on alveolar macrophages in idiopathic pulmonary fibrosis. Moreover, macrophages exhibited distinct differences in shape with actin-rich protrusions on fibrous collagen and more filopodia on globular collagen. In addition to these cytoskeletal changes, transmigration was higher when macrophages were cultured on fibrous collagen. Together these findings indicate that macrophages are sensitive to collagen morphology, responding with subtle changes in marker expression, shape and behavior rather than a complete polarization switch. This study emphasizes the complex interaction between macrophages and their surroundings, and the need for further exploration of both mechanical and morphological aspects.

Invadopodia Formation is a Critical Step in Cancer Cell Invasion: The Effect of Passage Number on Invadopodia Formation in MDA-MB-231 Breast Cancer Cell Line

High invasive cancer cells are thought to recruit specialised actin-rich protrusions for invasion in metastasis process. These protrusions are termed invadopodia. To study invadopodia formation, one of the first challenges faced by researchers has been to optimise the cell line passage number in order to be used for the invadopodia assay. Therefore, this study aims to investigate the effects of the passage number on invadopodia formation in MDA-MB-231 breast cancer cell line. Invadopodia assay was used to achieve the aim of the study. The results provided evidence that invadopodia formation is affected by the high passage number. The cells were also tested with dimethyloxalylglycine (DMOG) a hypoxic mimicking agent which is known to be an invadopodia inducer, the results showed that the cells in low passage number (P7) treated with DMOG increase the cells forming invadopodia, while the cells with high passage number (P35) showed that DMOG fails to stimulate the cells to form invadopodia. Furthermore, the cells with high passage number after passage 15 are starting to lose the ability to degrade the gelatin. In conclusion, this study suggests that only cells with a low passage number, less than passage 15 should be used in the study of invadopodia formation to obtain the results in the search for molecular targets and signaling at invadopodia.

STIM1-dependent Ca2+ signaling regulates podosome formation to facilitate cancer cell invasion

The clinical significance of STIM proteins and Orai Ca2+ channels in tumor progression has been demonstrated in different types of cancers. Podosomes are dynamic actin-rich cellular protrusions that facilitate cancer cell invasiveness by degrading extracellular matrix. Whether STIM1-dependent Ca2+ signaling facilitates cancer cell invasion through affecting podosome formation remains unclear. Here we show that the invasive fronts of cancer tissues overexpress STIM1, accompanied by active store-operated Ca2+ entry (SOCE). Interfering SOCE activity by SOCE inhibitors and STIM1 or Orai1 knockdown remarkably affects podosome rosettes formation. Mechanistically, STIM1-silencing significantly alters the podosome rosettes dynamics, shortens the maintenance phase of podosome rosettes and reduces cell invasiveness. The subsequently transient expression of STIM1 cDNA in STIM1-null (STIM1−/−) mouse embryo fibroblasts rescues the suppression of podosome formation, suggesting that STIM1-mediated SOCE activation directly regulates podosome formation. This study uncovers SOCE-mediated Ca2+ microdomain that is the molecular basis for Ca2+ sensitivity controlling podosome formation.

β3 integrin expression is required for invadopodia-mediated ECM degradation in lung carcinoma cells.

Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether β3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-β induction of β3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-β exposure. Invadopodia formation and degradation activity is dependent on β3 integrin expression since β3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in β3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for β3 integrin in invadopodia. Our results suggest that β3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. β3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.

The Invading Anchor Cell Induces Lateral Membrane Constriction during Vulval Lumen Morphogenesis in C. elegans

During epithelial tube morphogenesis, linear arrays of cells are converted into tubular structures through actomyosin-generated intracellular forces that induce tissue invagination and lumen formation. We have investigated lumen morphogenesis in the C.elegans vulva. The first discernible event initiating lumen formation is the apical constriction of the two innermost primary cells (VulF). The VulF cells thereafter constrict their lateral membranes along the apicobasal axis to extend the lumen dorsally. Lateral, but not apical, VulF constriction requires the prior invasion of the anchor cell (AC). The invading AC extends actin-rich protrusions toward VulF, resulting in the formation of a direct AC-VulF interface. The recruitment of the F-BAR-domain protein TOCA-1 to the AC-VulF interface induces the accumulation of force-generating actomyosin, causing a switch from apical to lateral membrane constriction and the dorsal extension of the lumen. Invasive cells may induce shape changes in adjacent cells to penetrate their target tissues.

Differential Depth Sensing Reduces Cancer Cell Proliferation via Rho-Rac-Regulated Invadopodia.

Bone, which is composed of a porous matrix, is one of the principal secondary locations for cancer. However, little is known about the effect of this porous microenvironment in regulating cancer cell proliferation. Here, we examine how the depth of the pores can transduce a mechanical signal and reduce the proliferation of noncancer breast epithelial cells (MCF-10A) and malignant breast cancer cells (MDA-MB-231 and MCF-7) using micrometer-scale topographic features. Interestingly, cells extend actin-rich protrusions, such as invadopodia, to sense the depth of the matrix pore and activate actomyosin contractility to decrease MCF-10A proliferation. However, in MDA-MB-231, depth sensing inactivates Rho-Rac-regulated actomyosin contractility and phospho-ERK signaling. Inhibiting contractility on this porous matrix using blebbistatin further reduces MDA-MB-231 proliferation. Our findings support the notion of mechanically induced dormancy through depth sensing, where invadopodia-mediated depth sensing can inhibit the proliferation of noncancer and malignant breast cancer cells through differential regulation of actomyosin contractility.

Cell adhesion controlled by adhesion G protein–coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo–Dock

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

Abstract 895: Fucosylation inhibits invadopodia formation and melanoma invasion

Abstract Background: Melanoma is the most deadly skin cancer and metastasis is essentially responsible for all melanoma related death. Recently, fucose treatment has been shown to inhibit melanoma cell migration and invasion in vitro, and melanoma metastasis in a mouse model, suggesting that protein fucosylation could be exploited to prevent or suppress melanoma metastasis. Invadopodia are proteolytic actin-rich protrusions used by metastatic melanoma cells to degrade extracellular matrix and to promote invasion and metastasis. In this study, we investigated the mechanisms by which fucosylation regulate melanoma invasion and invadopodia formation Method: The melanoma cells were treated with L-fucose-supplemented medium or infected with fucokinase-expressing lentivirus to increase protein fucosylation. shRNAs were also employed to knockdown fucokinase and to inhibit fucosylation in melanoma cells. The effects of fucosylation on invadopodia formation and extracellular matrix degradation were determined by phalloidin staining and degradation of fluorescently labeled gelatin thin film. Results: Here, we show that the addition of L-fucose to complete media inhibits the ability of melanoma cells to form invadopodia in a dose-dependent manner. The overexpression of hFUK in WM793 cells showed a similar result as L-fucose treatment, where invadopodia formation was significantly reduced. In addition, we observed a significant decrease in the percentage of cells that have invadopodia during L-fucose supplementation or hFUK overexpression. In an invadopodia precursor assay, the hFUK-overexpressing cell line exhibited delayed FBS-stimulated invadopodia formation. Conclusions: Our data suggest that fucose or the overexpression of hFUK reduces melanoma invasion by inhibiting invadopodia formation. Given that invadopodia formation was hindered within an hour after stimulation, we believe that invadopodia initiation is affected by increased fucosylation, resulting in fewer invadopodia and slower extracellular matrix degradation. Citation Format: Tyler S. Keeley, Eric Lau, Shengyu Yang. Fucosylation inhibits invadopodia formation and melanoma invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 895. doi:10.1158/1538-7445.AM2017-895

New waves in dendritic spine actin cytoskeleton: From branches and bundles to rings, from actin binding proteins to post-translational modifications.

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the number or strength of synapses are physiological mechanisms behind learning. The growth and maturation of dendritic spines and the activity-induced changes to their morphology are all based on changes to the actin cytoskeleton. In this review, we will discuss the regulation of the actin cytoskeleton in dendritic spine formation and maturation, as well as in synaptic strengthening. Concerning spine formation, we will focus on spine initiation, which has received less attention in the literature. We will also examine the recently revealed regulation of the actin cytoskeleton through post-translational modifications of actin monomers, in addition to the conventional regulation of actin via actin-binding proteins.

Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells.

Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation-promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor-Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient matrix degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, matrix degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces matrix degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.