Publications by authors named "Roberto Buccione"

38 Publications

Mineralogy and heavy metal assessment of the Pietra del Pertusillo reservoir sediments (Southern Italy).

Environ Sci Pollut Res Int 2021 Jan 19;28(4):4857-4878. Epub 2020 Sep 19.

Department of Sciences, University of Basilicata, viale dell'Ateneo Lucano 10, 8500, Potenza, Italy.

The Pietra del Pertusillo freshwater reservoir is a major artificial lake of environmental, biological, and ecological importance located in the Basilicata region, southern Italy. The reservoir arch-gravity dam was completed in 1963 for producing hydroelectric energy and providing water for human use, and nearby there are potential sources of anthropogenic pollution such as urban and industrial activities. For the first time, the minero-chemistry of the lake and fluvio-lacustrine sediments of the reservoir have been evaluated to assess the environmental quality. Moreover, the composition of fluvial sediments derived from the peri-lacual zone of the reservoir and of local outcropping bedrock were also studied to understand the factors affecting the behavior of elements in the freshwater reservoir, with particular attention paid to heavy metals. In Italy, specific regulatory values concerning the element threshold concentration for lake and river sediments do not exist, and for this reason, soil threshold values are considered the standard for sediments of internal waters. The evaluation of the environmental quality of reservoir sediments has been performed using enrichment factors obtained with respect to the average composition of a reconstructed local upper continental crust. We suggest this method as an innovative standard in similar conditions worldwide. In the studied reservoir sediments, the trace elements that may be of some environmental concern are Cr, Cu, Zn, As, and Pb although, at this stage, the distribution of these elements appears to be mostly driven by geogenic processes. However, within the frame of the assessment and the preservation of the quality of aquatic environments, particular attention has to be paid to As (which shows median value of 10 ppm, reaching a maximum value of 26 ppm in Quaternary sediments), constantly enriched in the lacustrine samples and especially in the fine-grained fraction (median = 8.5 ppm).
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http://dx.doi.org/10.1007/s11356-020-10829-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835177PMC
January 2021

A discussion on cell therapy in Manchester.

Stem Cell Res 2016 05 21;16(3):614-6. Epub 2016 Mar 21.

Centre for Regenerative Medicine "Stefano Ferrari", University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.scr.2016.03.005DOI Listing
May 2016

A Golgi-based KDELR-dependent signalling pathway controls extracellular matrix degradation.

Oncotarget 2015 Feb;6(5):3375-93

Unit of Genomic Approaches to Membrane Traffic, Fondazione Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy.

We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413660PMC
http://dx.doi.org/10.18632/oncotarget.3270DOI Listing
February 2015

Cytosolic phospholipase A₂ε drives recycling through the clathrin-independent endocytic route.

J Cell Sci 2014 Mar 10;127(Pt 5):977-93. Epub 2014 Jan 10.

Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy.

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.
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http://dx.doi.org/10.1242/jcs.136598DOI Listing
March 2014

EMBO molecular medicine: fast forward.

EMBO Mol Med 2014 01;6(1)

Institute of Cardiovascular Regeneration Centre of Molecular Medicine Goethe University, Frankfurt, Germany.

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http://dx.doi.org/10.1002/emmm.201303745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936492PMC
January 2014

Invasive cells in animals and plants: searching for LECA machineries in later eukaryotic life.

Biol Direct 2013 Apr 4;8. Epub 2013 Apr 4.

Department of Cell Biology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 43, Prague 2, Czech Republic.

Unlabelled: Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants - two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA.

Reviewer: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia.
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http://dx.doi.org/10.1186/1745-6150-8-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663805PMC
April 2013

Intracellular NAD(H) levels control motility and invasion of glioma cells.

Cell Mol Life Sci 2013 Jun 10;70(12):2175-90. Epub 2013 Jan 10.

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.
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http://dx.doi.org/10.1007/s00018-012-1249-1DOI Listing
June 2013

Cancer cell metabolism regulates extracellular matrix degradation by invadopodia.

Eur J Cell Biol 2013 Mar 8;92(3):113-21. Epub 2013 Jan 8.

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Medical Centre, Nijmegen, The Netherlands.

Transformed cancer cells have an altered metabolism, characterized by a shift towards aerobic glycolysis, referred to as 'the Warburg phenotype'. A change in flux through mitochondrial OXPHOS and cytosolic pathways for ATP production and a gain of capacity for biomass production in order to sustain the needs for altered growth and morphodynamics are typically involved in this global rewiring of cancer cell metabolism. Characteristically, these changes in metabolism are accompanied by enhanced uptake of nutrients like glucose and glutamine. Here we focus on the relationship between cell metabolism and cell dynamics, in particular the formation and function of invadopodia, specialized structures for focal degradation of the extracellular matrix. Since we recently found presence of enzymes that are active in glycolysis and associated pathways in invadopodia, we hypothesize that metabolic adaptation and invadopodia formation are linked processes. We give an overview on the background for this idea and show for the first time that extracellular matrix degradation by invadopodia can be differentially manipulated, without effects on cell proliferation, by use of metabolic inhibitors or changes in nutrient composition of cell culture media. We conclude that cell metabolism and carbohydrate availability, especially pyruvate, are involved in fuelling of invadopodia formation and activity.
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http://dx.doi.org/10.1016/j.ejcb.2012.11.003DOI Listing
March 2013

FGD1 as a central regulator of extracellular matrix remodelling--lessons from faciogenital dysplasia.

J Cell Sci 2012 Jul 1;125(Pt 14):3265-70. Epub 2012 Aug 1.

Université de Bordeaux, Physiopathologie du Cancer du Foie, U1053, F-33000 Bordeaux, France.

Disabling mutations in the FGD1 gene cause faciogenital dysplasia (also known as Aarskog-Scott syndrome), a human X-linked developmental disorder that results in disproportionately short stature, facial, skeletal and urogenital anomalies, and in a number of cases, mild mental retardation. FGD1 encodes the guanine nucleotide exchange factor FGD1, which is specific for the Rho GTPase cell division cycle 42 (CDC42). CDC42 controls cytoskeleton-dependent membrane rearrangements, transcriptional activation, secretory membrane trafficking, G1 transition during the cell cycle and tumorigenic transformation. The cellular mechanisms by which FGD1 mutations lead to the hallmark skeletal deformations of faciogenital dysplasia remain unclear, but the pathology of the disease, as well as some recent discoveries, clearly show that the protein is involved in the regulation of bone development. Two recent studies unveiled new potential functions of FGD1, in particular, its involvement in the regulation of the formation and function of invadopodia and podosomes, which are cellular structures devoted to degradation of the extracellular matrix in tumour and endothelial cells. Here, we discuss the hypothesis that FGD1 might be an important regulator of events controlling extracellular matrix remodelling and possibly cell invasion in physiological and pathological settings. Additionally, we focus on how studying the cell biology of FGD1 might help us to connect the dots that link CDC42 signalling with remodelling of the extracellular matrix (ECM) in physiology and complex diseases, while, at the same time, furthering our understanding of the pathogenesis of faciogenital dysplasia.
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http://dx.doi.org/10.1242/jcs.093419DOI Listing
July 2012

PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1.

PLoS One 2012 11;7(6):e37352. Epub 2012 Jun 11.

Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy.

Background: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose) polymerase (PARP/ARTD) family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain.

Methodology/principal Findings: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme.

Conclusions/significance: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037352PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372510PMC
November 2012

NG2-mediated Rho activation promotes amoeboid invasiveness of cancer cells.

Eur J Cell Biol 2012 Nov-Dec;91(11-12):969-77. Epub 2012 Jun 13.

Department of Cell Biology, Faculty of Science, Charles University in Prague, Czech Republic.

The aim of this study was to analyze the potential role of NG2 chondroitin sulfate proteoglycan in amoeboid morphology and invasiveness of cancer cells. In the highly metastatic amoeboid cell lines A3 and A375M2, siRNA-mediated down-regulation of NG2 induced an amoeboid-mesenchymal transition associated with decreased invasiveness in 3D collagen and inactivation of the GTPase Rho. Conversely, the expression of NG2 in mesenchymal sarcoma K2 cells as well as in A375M2 cells resulted in an enhanced amoeboid phenotype associated with increased invasiveness and elevated Rho-GTP levels. Remarkably, the amoeboid-mesenchymal transition in A375M2 cells triggered by NG2 down-regulation was associated with increased extracellular matrix-degrading ability, although this was not sufficient to compensate for the decreased invasive capability caused by down-regulated Rho/ROCK signaling. Conversely, in K2 cells with overexpression of NG2, the ability to degrade the extracellular matrix was greatly reduced. Taken together, we suggest that NG2-mediated activation of Rho leading to effective amoeboid invasiveness is a possible mechanism through which NG2 could contribute to tumor cell invasion and metastasis.
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http://dx.doi.org/10.1016/j.ejcb.2012.05.001DOI Listing
March 2013

Filamin A controls matrix metalloproteinase activity and regulates cell invasion in human fibrosarcoma cells.

J Cell Sci 2012 Aug 17;125(Pt 16):3858-69. Epub 2012 May 17.

Department of Pharmacology, Department of Cell Biology and Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, CT 06520-8066, USA.

Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered filamin expression or filamin mutations.
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http://dx.doi.org/10.1242/jcs.104018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462082PMC
August 2012

Polarised apical-like intracellular sorting and trafficking regulates invadopodia formation and degradation of the extracellular matrix in cancer cells.

Eur J Cell Biol 2012 Nov-Dec;91(11-12):961-8. Epub 2012 May 6.

Tumour Cell Invasion Laboratory, Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti, Italy.

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities. We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes. To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation. Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes.
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http://dx.doi.org/10.1016/j.ejcb.2012.03.005DOI Listing
March 2013

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

Toxicol Appl Pharmacol 2012 Apr 17;260(2):155-61. Epub 2012 Feb 17.

Department of Health Sciences, University of L'Aquila, Via Vetoio, L'Aquila, Italy.

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.
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http://dx.doi.org/10.1016/j.taap.2012.02.005DOI Listing
April 2012

The Aarskog-Scott syndrome protein Fgd1 regulates podosome formation and extracellular matrix remodeling in transforming growth factor β-stimulated aortic endothelial cells.

Mol Cell Biol 2011 Nov 12;31(22):4430-41. Epub 2011 Sep 12.

Université de Bordeaux, Physiopathologie du Cancer du Foie, U1053, F-33000 Bordeaux, France.

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.
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http://dx.doi.org/10.1128/MCB.05474-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209245PMC
November 2011

Effects of trifluralin on the mouse ovary.

Environ Toxicol 2013 Apr 4;28(4):201-6. Epub 2011 May 4.

Dipartimento di Scienze della Salute, Università degli Studi dell'Aquila, L'Aquila, Italy.

Trifluralin, a herbicide used to protect many arable and horticultural crops, was evaluated for its potential toxicity on the mammalian ovary. To this end, adult female mice were fed or not (control) with a trifluralin-enriched diet (150 mg/kg body weight/day) during gestation and lactation. After weaning, 3-week-old female mice from either trifluralin-treated or control groups were used to evaluate whether the exposure to this herbicide in utero and during lactation could induce stress responses in the ovary. It was found that trifluralin exposure caused a significantly higher level of p53, but not of pRb, in the whole ovary, and in particular in granulosa cells. TUNEL staining showed that herbicide treatment did not increase the apoptotic index of the somatic compartment. Also oocyte fertilizability was unaffected, as metaphase II oocytes retrieved from treated mice were capable of forming male and female pronuclei after in vitro fertilization as control mice. However, trifluralin determined a slightly higher number of oocytes with cytoplasmic degeneration compared with control animals. In conclusion, our results suggest that exposure to a low trifluralin dose during pregnancy and lactation does not impair oocyte quality, but can induce a stress response in ovarian somatic cells.
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http://dx.doi.org/10.1002/tox.20711DOI Listing
April 2013

Novel invadopodia components revealed by differential proteomic analysis.

Eur J Cell Biol 2011 Feb-Mar;90(2-3):115-27. Epub 2010 Jul 6.

Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti 66030, Italy.

When highly invasive cancer cells are cultured on an extracellular matrix substrate, they extend proteolytically active membrane protrusions, termed invadopodia, from their ventral surface into the underlying matrix. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete. To accelerate component discovery, we resorted to a proteomics approach by applying DIfference Gel Electrophoresis (DIGE) to compare invadopodia-enriched sub-cellular fractions with cytosol and cell body membrane fractions and the whole cell lysate. The fractionation procedure was validated through step-by-step monitoring of the enrichment in typical actin-related invadopodia-associated proteins. After statistical analysis, 129 protein spots were selected for peptide mass fingerprinting analysis; of these 76 were successfully identified and found to correspond to 58 proteins belonging to different functional classes including aerobic glycolysis and other metabolic pathways, protein synthesis, degradation and folding, cytoskeletal components and membrane-associated proteins. Finally, validation of a number of identified proteins was carried out by a combination of immuno-blotting on cell fractions and immunofluorescence localization at invadopodia. These results reveal newly identified components of invadopodia and open further avenues to the molecular study of invasive growth behavior of cancer cells.
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http://dx.doi.org/10.1016/j.ejcb.2010.05.004DOI Listing
July 2011

Aiming for invadopodia: organizing polarized delivery at sites of invasion.

Trends Cell Biol 2010 Feb 20;20(2):64-70. Epub 2009 Nov 20.

Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, S. Maria Imbaro (Chieti), 66030 Italy.

Recent years have witnessed growing interest in the biology of invadopodia, proteolytically active protrusions formed by invasive tumor cells when cultured on an extracellular matrix (ECM). Although substantial progress has been made towards defining their basic elements and features, the need remains to understand how these components are recruited and, ultimately, how ECM degradation is so precisely localized. According to recent evidence, invadopodia are raft-like membrane domains where cholesterol levels are tightly regulated, and active transport of protease-delivering carriers is required for their function. On this basis we hypothesize that the correct delivery of cargo to invadopodia is ensured by a polarized, cholesterol-dependent trafficking mechanism, similar to that of the apical domain of epithelial cells.
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http://dx.doi.org/10.1016/j.tcb.2009.10.006DOI Listing
February 2010

Caveolae mediate growth factor-induced disassembly of adherens junctions to support tumor cell dissociation.

Mol Biol Cell 2009 Oct 29;20(19):4140-52. Epub 2009 Jul 29.

Mayo Clinic, Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Rochester, MN 55905, USA.

Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.
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http://dx.doi.org/10.1091/mbc.e08-10-1043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754928PMC
October 2009

Cell and molecular biology of invadopodia.

Int Rev Cell Mol Biol 2009 ;275:1-34

Tumor Cell Invasion Laboratory, Chieti, Italy.

The controlled degradation of the extracellular matrix is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years toward understanding the basic molecular components and their ultrastructural features; generating substantial interest in invadopodia as a paradigm to study the complex interactions between the intracellular trafficking, signal transduction, and cytoskeleton regulation machineries. The next level will be to understand whether they may also represent valid biological targets to help advance the anticancer drug discovery process. Current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology.
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http://dx.doi.org/10.1016/S1937-6448(09)75001-4DOI Listing
August 2009

Faciogenital dysplasia protein (FGD1) regulates export of cargo proteins from the golgi complex via Cdc42 activation.

Mol Biol Cell 2009 May 4;20(9):2413-27. Epub 2009 Mar 4.

Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy.

Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY). FGD1 encodes a guanine nucleotide exchange factor that specifically activates the GTPase Cdc42. In turn, Cdc42 is an important regulator of membrane trafficking, although little is known about FGD1 involvement in this process. During development, FGD1 is highly expressed during bone growth and mineralization, and therefore a lack of the functional protein leads to a severe phenotype. Whether the secretion of proteins, which is a process essential for bone formation, is altered by mutations in FGD1 is of great interest. We initially show here that FGD1 is preferentially associated with the trans-Golgi network (TGN), suggesting its involvement in export of proteins from the Golgi. Indeed, expression of a dominant-negative FGD1 mutant and RNA interference of FGD1 both resulted in a reduction in post-Golgi transport of various cargoes (including bone-specific proteins in osteoblasts). Live-cell imaging reveals that formation of post-Golgi transport intermediates directed to the cell surface is inhibited in FGD1-deficient cells, apparently due to an impairment of TGN membrane extension along microtubules. These effects depend on FGD1 regulation of Cdc42 activation and its association with the Golgi membranes, and they may contribute to FGDY pathogenesis.
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http://dx.doi.org/10.1091/mbc.e08-11-1136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675621PMC
May 2009

Invadopodia biogenesis is regulated by caveolin-mediated modulation of membrane cholesterol levels.

J Cell Mol Med 2009 Aug 31;13(8B):1728-1740. Epub 2008 Oct 31.

Tumor Cell Invasion Laboratory, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Chieti, Italy.

Invadopodia are proteolytically active protrusions formed by invasive tumoural cells when grown on an extracellular matrix (ECM) substratum. Clearly, invadopodia are specialized membrane domains acting as sites of signal transduction and polarized delivery of components required for focalized ECM degradation. For these reasons, invadopodia are a model to study focal ECM degradation by tumour cells. We investigated the features of invadopodia membrane domains and how altering their composition would affect invadopodia biogenesis and function. This was achieved through multiple approaches including manipulation of the levels of cholesterol and other lipids at the plasma membrane, alteration of cholesterol trafficking by acting on caveolin 1 expression and phosphorylation. We show that cholesterol depletion impairs invadopodia formation and persistence, and that invadopodia themselves are cholesterol-rich membranes. Furthermore, the inhibition of invadopodia formation and ECM degradation after caveolin 1 knock-down was efficiently reverted by simple provision of cholesterol. In addition, the inhibitory effect of caveolin 3(DGV) expression, a mutant known to block cholesterol transport to the plasma membrane, was similarly reverted by provision of cholesterol. We suggest that invadopodia biogenesis, function and structural integrity rely on appropriate levels of plasma membrane cholesterol, and that invadopodia display the properties of cholesterol-rich membranes. Also, caveolin 1 exerts its function in invadopodia formation by regulating cholesterol balance at the plasma membrane. These findings support the connection between cholesterol, cancer and caveolin 1, provide further understanding of the role of cholesterol in cancer progression and suggest a mechanistic framework for the proposed anti-cancer activity of statins, tightly related to their blood cholesterol-lowering properties.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00568.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512369PMC
August 2009

Invadopodia: specialized tumor cell structures for the focal degradation of the extracellular matrix.

Cancer Metastasis Rev 2009 Jun;28(1-2):137-49

Department of Cell Biology and Oncology, Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, S. Maria Imbaro, (Chieti), 66030, Italy.

Invasive tumor-derived or transformed cells, cultured on a flat extracellular matrix substratum, extend specialized proteolytically active plasma membrane protrusions. These structures, termed invadopodia, are responsible for the focal degradation of the underlying substrate. Considerable progress has been made in recent years towards understanding the basic molecular components and regulatory circuits and the ultrastructural features of invadopodia. This has generated substantial interest in invadopodia as a paradigm to study the complex interactions between the intracellular trafficking, signal transduction and cytoskeleton regulation machineries; hopes are high that they may also represent valid biological targets to help advance the anti-cancer drug discovery process. Current knowledge will be reviewed here with an emphasis on the many open questions in invadopodia biology.
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http://dx.doi.org/10.1007/s10555-008-9176-1DOI Listing
June 2009

Faciogenital dysplasia protein Fgd1 regulates invadopodia biogenesis and extracellular matrix degradation and is up-regulated in prostate and breast cancer.

Cancer Res 2009 Feb 13;69(3):747-52. Epub 2009 Jan 13.

Department of Cell Biology and Oncology, Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, S Maria Imbaro, Chieti, Italy.

Invadopodia are proteolytically active membrane protrusions that extend from the ventral surface of invasive tumoral cells grown on an extracellular matrix (ECM). The core machinery controlling invadopodia biogenesis is regulated by the Rho GTPase Cdc42. To understand the upstream events regulating invadopodia biogenesis, we investigated the role of Fgd1, a Cdc42-specific guanine nucleotide exchange factor. Loss of Fgd1 causes the rare inherited human developmental disease faciogenital dysplasia. Here, we show that Fgd1 is required for invadopodia biogenesis and ECM degradation in an invasive cell model and functions by modulation of Cdc42 activation. We also find that Fgd1 is expressed in human prostate and breast cancer as opposed to normal tissue and that expression levels matched tumor aggressiveness. Our findings suggest a central role for Fgd1 in the focal degradation of the ECM in vitro and, for the first time, show a connection between Fgd1 and cancer progression, proposing that it might function during tumorigenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-1980DOI Listing
February 2009

Assembly and biological role of podosomes and invadopodia.

Curr Opin Cell Biol 2008 Apr 11;20(2):235-41. Epub 2008 Mar 11.

Unit of Actin Cytoskeleton Regulation, Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, Via Nazionale 8a, 66030 Santa Maria Imbaro Chieti, Italy.

Regulated tissue invasion via motile and lytic events is critical for physiological processes such as immune system function and inflammatory responses, wound healing, and organ development, but pathological subversion of this process drives tumour cell invasion and metastasis. Cell migration and invasion require the integration of several processes that include: first, the local modulation of cytoskeleton structure and contractile forces; second, the turnover of substrate adhesions and their associated microfilaments; and third, the generation of specialised, transient domains that mediate the protease-dependent focal degradation of the extracellular matrix. Recent work has re-discovered prominent actin-based cellular structures, termed invadopodia and podosomes, as unique structural and functional modules through which major invasive mechanisms are regulated. The stage is now set to unravel their roles in the physiology and pathology of tissue plasticity and repair.
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http://dx.doi.org/10.1016/j.ceb.2008.01.005DOI Listing
April 2008

Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation.

J Cell Sci 2008 Feb 15;121(Pt 3):369-78. Epub 2008 Jan 15.

Tumour Cell Invasion Laboratory, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, S. Maria Imbaro (Chieti), Italy.

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. Although many molecular components have been defined, less is known of the formation and regulation of invadopodia. The multidomain protein cortactin, which is involved in the regulation of actin polymerisation, is one such component, but how cortactin is modulated to control the formation of invadopodia has not been elucidated. Here, a new invadopodia synchronization protocol is used to show that the cortactin N-terminal acidic and SH3 domains, involved in Arp2/3 complex and N-WASP binding and activation, respectively, are both required for invadopodia biogenesis. In addition, through a combination of RNA interference and a wide array of cortactin phosphorylation mutants, we were able to show that three convergent regulatory inputs based on the regulation of cortactin phosphorylation by Src-family kinases, Erk1/Erk2 and PAK are necessary for invadopodia formation and extracellular matrix degradation. These findings suggest that cortactin is a scaffold protein bringing together the different components necessary for the formation of the invadopodia, and that a fine balance between different phosphorylation events induces subtle changes in structure to calibrate cortactin function.
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http://dx.doi.org/10.1242/jcs.008037DOI Listing
February 2008

Actin dynamics at sites of extracellular matrix degradation.

Eur J Cell Biol 2006 Dec 28;85(12):1217-31. Epub 2006 Sep 28.

Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8A, I-66030 S. Maria Imbaro, Chieti, Italy.

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.
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http://dx.doi.org/10.1016/j.ejcb.2006.08.003DOI Listing
December 2006

Adhesions that mediate invasion.

Int J Biochem Cell Biol 2006 17;38(11):1875-92. Epub 2006 May 17.

Unit of Actin Cytoskeleton Regulation, Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, Via Nazionale 8a, 66030 Santa Maria Imbaro, Italy.

Infiltration of new tissue areas requires that a mammalian cell overcomes the physical and biochemical barrier of the surrounding extracellular matrix. Cell migration during embryonic development, and growth, invasion and dispersal of metastatic tumor cells depend to a large extent on the controlled degradation of extracellular matrix components. Localized degradation of the surrounding matrix is seen at defined adhesive (podosomes) and/or protrusive (invadopodia) locations in a variety of normal cells and aggressive carcinoma cells, suggesting that these membrane-associated cellular devices have a central role in mediating polarized migration in cells that cross-tissue boundaries. Here, we will discuss the recent advances and developments in this field, and provide our provisional outlook into the future understanding of the principles of focal extracellular matrix degradation by podosomes and invadopodia.
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http://dx.doi.org/10.1016/j.biocel.2006.05.003DOI Listing
September 2006

Invadopodia: a guided tour.

Eur J Cell Biol 2006 Apr 11;85(3-4):159-64. Epub 2005 Oct 11.

Tumor Cell Invasion Unit, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, I-66030 S. Maria Imbaro (Chieti), Italy.

The controlled degradation of extracellular matrix is crucial in physiological and pathological cell invasion alike. In cultured cells, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years towards understanding the basic molecular components and the ultrastructural features of invadopodia. This current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology. Considering the substantial interest and momentum in the field, the need for an operational framework to correctly define and identify invadopodia will also be discussed.
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http://dx.doi.org/10.1016/j.ejcb.2005.09.005DOI Listing
April 2006