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    Archived pages: 107 . Archive date: 2014-09.

  • Title: Home
    Descriptive info: .. English.. Deutsch.. Topics.. Overview.. Training.. Research.. Participants.. Press and PR.. Publications.. Publications: explained.. Links.. Search.. Secure Area.. Cardiovascular and cancer-related diseases are the leading causes of human mortality and disability.. The underlying mechanisms originate from chronic interstitial cell activation leading to pathological tissue remodelling and malfunction of cells.. These depend on three fundamental processes: Cell adhesion, migration, and modulation/degradation of the extracellular matrix (ECM), which together determine tissue invasion and remodelling.. The.. Tissue Transmigration Training.. Network..  ...   technologies and complementary skills training.. T3Net has a multidisciplinary training approach and is thus crucial in bundling current and future European expertise and to consolidate the momentary European leadership in these emerging key areas of biomedical research.. T3Net aims to establish a long-term European network based on a new cohort of young professionals with the potential to exploit their knowledge in academic, clinical or industrial settings.. News.. no news in this list.. to the top.. Imprint.. 2010 T3-Net..

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  • Title: Home
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  • Title: Home
    Descriptive info: Übersicht.. Ausbildung.. Forschung.. Teilnehmer.. Presse und PR.. Veröffentlichungen.. Veröffentlichungen: leicht erklärt.. Suche.. Interner Bereich.. Überblick.. Kardiovaskuläre Defekte sowie Krebserkrankungen sind maßgebliche Ursachen für Invalidität und Tod.. Die zugrunde liegenden Mechanismen liegen in der chronischen Zellaktivierung, die zu einer pathologischen Veränderung des Gewebes und zu Funktionsstörungen von Zellen führt.. Diese hängen von drei wesentlichen Prozessen ab: Zelladhäsion, Migration und der Veränderung bzw.. dem Abbau der extrazellulären Matrix (EZM).. Zusammen bestimmen diese Faktoren die Invasion von Zellen in Gewebe und deren Umgestaltung.. Das.. Tissue Transmigration Training Network..  ...   auf dem Training in innovativen Technologien in Verbindung mit komplementären Fähigkeiten ( soft skills ).. T3Net bietet damit eine zeitgemäße multidisziplinäre Ausbildungsweise an und wird so entscheidend dazu beitragen, die Expertise in diesen Schlüsselgebieten der Biowissenschaften in Europa zu bündeln und die Europäische Führungsrolle in dieser Forschungsrichtung weiter zu festigen.. Ziele von T3Net sind der Auf- und Ausbau eines Europäischen Forschungsnetzwerks, sowie die Ausbildung junger Experten, die fähig sind, ihre Fachkenntnisse sowohl in einer akademischen, klinischen oder industriellen Umgebung anzuwenden.. Keine Artikel in dieser Ansicht.. Impressum..

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  • Title: Overview
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  • Title: Training
    Descriptive info: The objective of T3Net is to foster a group of young scientists with specialized training in integrating dynamic cell imaging techniques and high content screening approaches with the biology and pathophysiology underlying cardiovascular diseases and cancer.. The academic and industrial partners provide training in new areas of research including the cell structures mediating invasion such as podosomes and invadopodia, as well as the application of materials sciences, nanotechnology and state-of-the-art in vivo imaging.. These techniques are applied to study ECM-cell interactions, in models encompassing vascular remodelling, immunity, inflammation and bone physiology, as well as the pathophysiology  ...   and tissue transmigration in physiology and pathology.. They also benefit from valuable complementary skills training including communication,.. entrepreneurship.. and intellectual property rights.. Training is articulated in multiple layers of intervention:.. Local.. (training through individual research projects and secondments).. Institutional.. (seminar programmes, specific and general courses, etc.. ).. Network-wide activities.. (annual network symposia, organization of workshops/training courses and visits to partner laboratories and SMEs for scientific collaborations and courses).. An additional innovative feature is to provide high quality complementary skills training to all fellows through an academic associated partner: Open University, UK (.. http://www.. open.. ac.. uk/.. )..

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  • Title: Research
    Descriptive info: Research within T3Net is conducted in several work packages, which all focus on different cellular and molecular aspects of invasive cell migration.. 1) Cancer invasion and dissemination.. 1.. 1 Morpho-functional basis of invadopodia structure and function.. This task aims to unveil the morpho-functional basis of the highly organized structure and function of invadopodia in tumour cells.. The role of membrane lipids, particularly cholesterol and caveolin 1, will be studied through the manipulation of membrane lipid composition.. 2 Role of Fgd1 and podoplanin in linking ECM-cell interactions and formation of invadopodia.. This task will analyse the role of two key proteins, Fgd1 and.. podoplanin, mediating the signalling events linking extracellular matrix degrading (ECM)-cell interactions and the formation of.. invadopodia in tumor cells and podosomes in dendritic cells (DCs) and endothelial cells.. 2) Immunity, inflammation and vascular remodelling.. 2.. 1 TGF.. β.. receptors involved in podosome formation in endothelial cells.. Experiments will be performed aimed at identifying the TGF.. β.. receptor and co-receptor involved in podosome formation in endothelial cells and dissecting the molecular links to downstreameffectors in the process of endothelial podosome formation and ECM degradation.. 2 Regulation of WASP, a critical player in podosome formation in dendritic cells.. Regulation of WASP (Wiskott-Aldrich Syndrome Protein) by WASP-Interacting Protein (WIP) will be investigated, including the effect of WIP phosphorylation, regulation of actin filament assembly and control of WASP degradation.. 3 Intracellular transport processes in podosome regulation.. This.. project will investigate intracellular transport processes in podosome regulation, with particular emphasis on the role of vesicle-regulating proteins.. 4 Characterisation of proteinases involved in ECM degradation by podosomes.. This project aims to identify the specific proteinases involved in podosome-localized matrix degradation, their modes of transport to podosomes, and the mechanisms of their  ...   associated changes in ocal contractility.. 3 Structural characteristics of podosomes formed in vitro and in vivo.. The differing structural characteristics of podosomes formed under in vitro and in vivo conditions will be explored in detail, using immunofluorescence and confocal microscopy.. 3D reconstruction of serial images will allow visualization of the architectural organisation of the structures.. 4 Assembly of the sealing zone by osteoclasts.. In this project, the mechanisms will be addressed whereby osteoclasts interact with bone surfaces, sense chemical and physical features of the bone and assemble the resorptive sealing zone which can degrade bone.. 4) Unravelling the basic molecular machinery- the search for new components.. 4.. 1 Genes involved in regulation of podosome formation.. In this task, the expression of specific adhesion-associated and signalling molecules that might be involved in the regulation of podosome and invadopodia formation, will be suppressed using a siRNA approach.. 2 Comparative analysis of invadopodia and.. podosome-enriched cell fractions.. This two-pronged approach will.. allow for an in-depth comparative analysis between podosomes and invadopodia, which are similar yet unique structures in different cell models.. 3 Transcriptomic analysis of DC-derived osteoclasts.. This taskwill identify genes specifically implicated in bone resorption.. Selected proteins will also be validated in a 3D environment.. 5) The third dimension.. 5.. 1 Role of lytic protrusions in 3D tissue invasion.. These studies will show precisely how invadopodia contribute to 3D cell invasion and tissue structure remodelling.. 2 Matrix-degrading structures in additional models.. The approach in 5.. 1 will be extended to relevant 3D models of endothelial cell, macrophage and osteoclast podosomes.. 3 Investigation in human tumour samples.. A direct effort to detect lytic protrusive structures will be carried out in human tumour samples, using high-end microscopy and ECM degradation assays..

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  • Title: Participants
    Descriptive info: Fellows.. Supervisors.. Institutions.. Management.. (Learn more about recent T3Net trainees and their projects).. (Learn more about the scientists supervising T3Net trainees).. (Learn more about about the Institutions participating the T3Net).. (Learn more about the coordinators of the T3Net)..

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  • Title: Press and PR
    Descriptive info: Contact us.. The T3Net is very grateful for the public interest.. We welcome you to ask further questions and get more information by contacting our project management:.. Prof.. Dr.. Stefan Linder (Coordinator).. Universitätsklinikum Eppendorf.. Institut für Medizinische Mikrobiologe, Virologie und Hygiene.. Campus Forschung, Gebäude N27.. Martinistr.. 52.. 20246 Hamburg.. GERMANY.. Phone: +49 40 7410 55175.. Fax: +49 40 7410 54881.. Email:.. s.. linder@.. we dont want spam.. uke.. de.. Press Releases.. June 2013:.. Testing the limits of cell migration.. April  ...   insider perspective on the EU programme T3net.. April 2013:.. Cutting-Edge research in Hamburg.. (in German).. 2012:.. Chercheur d Aquitaine: Issue 2, published in 2012.. (English).. January 2012:.. Article in.. Cell News 01/2012.. Mai 2011:.. Chercheurs d'aquitaine.. (in French).. March 2010:.. Press Release in the magazine Laborjournal (German).. 13.. Jan 2010:.. Press Release of the Universitätsklinikum Hamburg-Eppendorf (German).. English version can be downloaded.. here.. January 2010:.. Press release in the scientific internet portal Bionity.. com (German).. Press release in Ad-Hoc News (German)..

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  • Title: Publications
    Descriptive info: Integrin-Matrix Clusters Form Podosome-like Adhesions in the Absence of Traction Forces.. Yu CH, Rafiq NB, Krishnasamy A, Hartman KL, Jones GE, Bershadsky AD, Sheetz MP.. Cell Rep.. 2013 Dec 12;5(5):1456-68.. doi: 10.. 1016/j.. celrep.. 2013.. 10.. 040.. Epub 2013 Nov 27.. Comparative transcriptomics reveal RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts.. Georgess D.. , Mazzorana M, Terrado J, Delprat C, Chamot C, Guasch RM, Pérez-Roger I,.. Jurdic P.. , Machuca-Gayet I.. Mol Biol Cell.. 2013 Nov 27.. [Epub ahead of print].. Preclinical intravital microscopy of the tumour-stroma interface: invasion, metastasis, and therapy response.. Alexander S, Weigelin B, Winkler F,.. Friedl P.. Curr Opin Cell Biol.. 2013 Oct;25(5):659-71.. ceb.. 07.. 001.. Epub 2013 Jul 26.. WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility.. Inmaculada Banon-Rodriguez,.. Julia Saez de Guinoa, Alejandra Bernardini, Chiara Ragazzini, Estefania Fernandez, Yolanda R.. Carrasco,.. Gareth E.. Jones.. , Francisco Wandosell, Ines Maria Anton.. PLoS ONE 8(8): e70364.. doi:10.. 1371/journal.. pone.. 0070364, August 7, 2013.. Podosome reformation in macrophages: assays and analysis.. Cervero P.. , Panzer L,.. Linder S.. Methods Mol Biol.. 2013;1046:97-121.. 1007/978-1-62703-538-5_6.. The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.. Godbout C, Follonier Castella L, Smith EA,.. Talele N.. , Chow ML, Garonna A,.. Hinz B.. PLoS One.. 2013 May 14;8(5):e64560.. 0064560.. Print 2013.. Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force.. Wolf K, Te Lindert M,.. Krause M.. , Alexander S, Te Riet J, Willis AL, Hoffman RM, Figdor CG, Weiss SJ,.. J Cell Biol.. 2013 Jun 24;201(7):1069-84.. 1083/jcb.. 201210152.. Drebrin preserves endothelial integrity by stabilizing nectin at adherens junctions.. Rehm K, Panzer L, van Vliet V,.. Genot E.. ,.. J Cell Sci.. 2013 Jun 7.. A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and 3D invasion of macrophages.. Wiesner C,.. Azzouzi KE.. 2013 Jul 1;126(Pt 13):2820-33.. WIP: WASP-interacting proteins at invadopodia and podosomes.. García E,.. Jones GE.. , Machesky LM, Antón IM.. Eur J Cell Biol.. 2012 Nov-Dec;91(11-12):869-77.. ejcb.. 2012.. 06.. 002.. Epub 2012 Jul 21.. The myofibroblast matrix:implications for tissue repair and fibrosis.. Klingberg F.. , White ES.. J Pathol.. 2012 Sep 21.. 1002/path.. 4104.. Proteomic analysis of podosome fractions from macrophages reveals similarities to spreading initiation centres.. , Himmel M, Krüger M,.. 2012 Nov;91(11-12):908-22.. 05.. 005.. Epub 2012 Jun 20.. Invadopodia and rolling-type motility are specific features of highly invasive p190(bcr-abl) leukemic cells.. Daubon T, Rochelle T, Bourmeyster N,.. Génot E.. 2012 Nov;91(11-12):978-87.. 04.. 006.. Epub 2012 Jun 19.. FGD1 as a central regulator of extracellular matrix remodelling--lessons from faciogenital dysplasia.. , Daubon T, Sorrentino V,.. Buccione R.. 2012 Jul 15;125(Pt 14):3265-70.. 1242/jcs.. 093419.. Epub 2012 Aug 1.. Mechanical aspects of lung fibrosis: a spotlight on the myofibroblast.. Proc Am Thorac Soc.. 2012 Jul;9(3):137-47.. 1513/pats.. 201202-017AW.. The role of the myofibroblast in tumor stroma.. remodeling.. Otranto M, Sarrazy V, Bonté F,.. , Gabbiani G, Desmoulière A.. Cell Adh Migr.. 2012 May-Jun;6(3):203-19.. 4161/cam.. 20377.. Epub 2012 May 1.. Filamin.. A controls matrix metalloprotease activity and regulates cell invasion in human fibrosarcoma cells.. Baldassarre M, Razinia Z, Brahme N,.. , Calderwood DA.. 2012 May 17.. Tyrosine phosphorylation of WASP promotes calpain-mediated podosome disassembly.. Macpherson L, Monypenny J, Blundell MP, Cory GO, Tomé-García J, Thrasher AJ,.. , Calle Y.. Haematologica.. 2012 May;97(5):687-91.. Epub 2011 Dec 1.. Polarised  ...   migration.. Katarina Wolf and.. Peter Friedl.. Trends Cell Biol.. 2011 Dec;21(12):736-44.. Epub 2011 Oct 27.. The Single-Molecule Mechanics of the Latent TGF-β1 Complex.. Buscemi L, Ramonet D,.. , Formey A, Smith-Clerc J, Meister JJ,.. Curr Biol.. 2011 Dec 6.. Substrate.. adhesion regulates sealing zone architecture and dynamics in cultured osteoclasts.. Anderegg F.. , Geblinger D, Horvath P, Charnley M, Textor M, Addadi L,.. Geiger B.. 2011;6(12):e28583.. Epub 2011 Dec 5.. Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.. Juin A, Billottet C, Moreau V, Destaing O, Albiges-Rizo C, Rosenbaum J,.. , Saltel F.. 2011 Nov 23.. Cancer invasion and the microenvironment: plasticity and reciprocity.. , Alexander S.. 2011 Nov 23;147(5):992-1009.. The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells.. Daubon T,.. Mol Cell Biol.. 2011 Nov;31(22):4430-41.. Epub 2011 Sep 12.. Interstitial guidance of cancer invasion.. Grytsenko P,.. Ilina O.. J Pathol.. 2011 Oct 18.. 3031.. Podosome rings generate forces that drive saltatory osteoclast migration.. Shiqiong Hu , Emmanuelle Planus ,.. Dan Georgess.. , Christophe Place , Xianghui Wang , Corinne Albiges-Rizo ,.. Pierre Jurdic.. , and Jean-Christophe Géminard.. 2011 Jul 7.. Novel invadopodia components revealed by differential proteomic analysis.. Francesca Attanasio, Giusi Caldieri, Giada Giacchettia, Remcovan Horssen, Bé Wieringa,.. Roberto Buccione.. , 2010.. European Journal of Cell Biology Vol.. 90, Issues 2-3, February-March 2011, Pages 115-127.. Invadosomes: Intriguing structures with promise.. Frédéric Saltel, Thomas Daubon, Amélie Juina,.. Isabel Egaña Ganuza.. , Véronique Veillat,.. 90, Issues 2-3, February-March 2011, Pages 100-107.. Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion.. Olga Ilina.. , Gert-Jan Bakker, Angela Vasaturo, Robert M Hofmann,.. , 2011.. Physical Biology Vol.. 8, Issue 1, February 2011.. Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.. Shieh AC, Rozansky HA,.. , Swartz MA.. Cancer Res.. 2011 Feb 1;71(3):790-800.. Epub 2011 Jan 18.. The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes.. Susanne Cornfine, Mirko Himmel, Petra Kopp,.. Karim el Azzouzi.. , Christiane Wiesner, Marcus Krüger, Thomas Rudel,.. Stefan Linder.. Molecular Biology of the Cell Vol.. 22, Issue 2, January 15, 2011, Pages 202-215.. Podoplanin associates with CD44 to promote directional cell migration.. Martín-Villar E, Fernández-Muñoz B, Parsons M, Yurrita MM, Megías D, Pérez-Gómez E,.. , Quintanilla M.. 2010 Dec;21(24):4387-99.. Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives.. , Gabbiani G.. F1000 Biol Rep.. 2010 Nov 11;2:78.. 3410/B2-78.. p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter.. Marcel V,.. Vijayakumar V.. , Fernández-Cuesta L, Hafsi H, Sagne C, Hautefeuille A, Olivier M, Hainaut P.. Oncogene.. 2010 May 6;29(18):2691-700.. 1038/onc.. 2010.. 26.. Epub 2010 Mar 1.. Regulation of myofibroblast activities: calcium pulls some strings behind the scene.. Follonier Castella L, Gabbiani G, McCulloch CA,.. Exp Cell Res.. 2010 Sep 10;316(15):2390-401.. Epub 2010 May 6.. The myofibroblast: paradigm for a mechanically active cell.. J Biomech.. 2010 Jan 5;43(1):146-55.. jbiomech.. 2009.. 09.. 020.. Epub 2009 Oct 3.. Hypoxia impairs skin myofibroblast differentiation and function.. Modarressi A, Pietramaggiori G, Godbout C, Vigato E, Pittet B,.. J Invest Dermatol.. 2010 Dec;130(12):2818-27.. 1038/jid.. 224.. Epub 2010 Aug 5.. A new lock-step mechanism of matrix remodelling based on subcellular contractile events.. Castella LF, Buscemi L, Godbout C, Meister JJ,.. 2010 May 15;123(Pt 10):1751-60.. Epub 2010 Apr 27..

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  • Title: Publications: explained
    Descriptive info: 2012 Jun 20.. Podosomes are multifunctional organelles of invasive cells, including immune cells such as macrophags.. Podosomes are cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing.. In this study, we aimed to identify all protein components that build up these important structures and also enable their multiple functions and regulate their turnover.. We used macrophages as a cell system, as these cells form numerous podosomes in the plane of adhesion, which makes them an ideal system for the purification of podosome-enriched cell fractions.. We combined isotopic labelling of cells with a differential lysis technique to gain podosome-enriched cell fractions.. Identification of protein components in these fractions was performed by mass spectrometry analysis.. We could identify.. 203 proteins, comprising 33 known podosome proteins and 170 potential novel components.. Software analyses show that particularly proteins involved in actin cytoskeleton regulation, adhesion mediation and those harboring ATPase or GTPase activity are enriched.. Surprisingly, we also identified a variety of ribosomal and RNA binding proteins.. This was corroborated by proof-of-principle experiments, in which the newly identified RNA binding protein hnRNP-K, and also the actin binding protein WDR1, could be identified as novel components of podosomes.. Our results point to the potential relevance of RNA binding proteins as additional regulators of podosome structure or function.. They also indicate that the list of identified proteins should be a useful and relevant source for the identification and study of novel podosome components and thus of novel regulators of macrophage adhesion and invasion in health and disease.. Figure legend:.. Generation of podosome-enriched cell fractions.. 3D reconstructions (.. upper images.. ) of human macrophages before (.. left.. ) or after differential cell lysis (.. right.. ), together with side views of the respective cells (.. lower images.. Cells.. are stained for podosomal proteins F-actin (red) and vinculin (green).. Note the dome-shaped part of the cell (.. left images.. ), which contains the nucleus and most of the cytoplasm, and the remaining adhesive part ( footplate ;.. right images.. ), which remains after lysis and contains podosomes.. White bars: 5 µm.. Cells of the myeloid lineage assemble characteristic highly dynamic actin-based adhesive structures termed podosomes that are thought to be involved in migration of cells that have to cross and invade tissue boundaries.. Podosomes cluster behind the extending leading edge of migrating myeloid cells and are required for polarization, persistent migration and chemotaxis.. We and others have identified the Arp2/3 activator Wiskott Aldrich Syndrome Protein (WASP) and the WASP interacting protein(WIP) in the podosome actin core.. where they play a major role in their formation and dynamics.. We have previously shown that the rapid turnover of podosomes of migrating myeloid cells involves cleavage of WASP by the protease calpain, supporting a role for WASP in both podosome formation and disassembly.. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown.. In this publication we show that tyrosine phosphorylation of human (Y291) and murine (Y293) WASP not only promotes a sustained WASP open conformation required for Arp2/3 binding and actin filament nucleation, but also enhances its susceptibility to calpain-mediated cleavage, thereby promoting disassembly of podosomes as the leading edge of the cell progresses forward.. Figure legend:.. The f-actin core of podosomes (red) are less stable and have shorter half lives when generated through human phosphomimic (Y291E) WASP (B) and phophodead (Y291F) WASP (C) generates very stable podosomes.. Addition of a calpain inhibitor (ALLM) restores the stability of Y291E-generated podosomes (E).. Podosomes are actin-rich adhesion and invasion structures.. Especially in macrophages, podosomes exist in two subpopulations: large precursors at the cell periphery and smaller podosomes, called successors, in the cell interior.. To date, the mechanisms that differentially regulate these subpopulations are largely unknown.. In this study, we could show that podosome subpopulations differ in their molecular composition.. We identified the membrane-associated protein supervillin as the first factor that localizes differentially to successor podosomes, and especially to a newly described cap structure that decorates the top of podosomes.. We further show that supervillin-mediated coupling of myosin-dependent contractility to podosomes is required for the regulation of podosome dynamics.. Consistently, depletion of supervillin from cells resulted in longer life times of podosomes.. We further show that supervillin also regulates cell polarization, which is a prerequisite for successful migration.. Supervillin is therefore involved in multiple aspects of macrophage adhesion, migration and invasion, which identifies it as an important factor for immune cell regulation in health and disease.. Supervillin localizes to a subpopulation of podosomes.. Confocal micrographs of primary human macrophages expressing fluorescently labelled supervillin (green) and stained for F-actin (red) to label podosomes.. Note that supervillin localizes to smaller successor podosomes in the inner part of a round, unpolarized cell (.. ) or to the rear of a polarized cell (.. Larger podosomes at the cell periphery (.. ) or the cell front (.. ) do mostly not contain supervillin.. Upper images: ca.. 30 µm, lower images: ca.. 40 µm.. Rheumatoid arthritis is the most severe inflammatory disease and affects 1% of the population worldwide.. Increasing evidence describes autoimmunity as the underlying cause of RA-induced bone loss.. Indeed, autoantibodies against citrullinated proteins (ACPAs) are among the strongest risk factors for bone destruction and are highly present in serum years before the onset of clinically overt RA.. Citrullination is a posttranslational modification of the arginine aminoacid into citrullin.. Mechanisms though which ACPAs effect, directly or indirectly, bone homeostasis are still unknown.. In this study, we found a significant association in RA-patients between ACPAs and serum markers for osteoclast (OC)-mediated bone resorption.. Also, we showed that OCs expressed enzymes that citrullinate proteins and thus giving new targets for ACPAs.. In particular, vimentin, a intermediate filament protein, was found to be highly citrullinated in differentiating OCs.. We therefore purified an antibody against this mutated citrullinated vimentin (MCV-ACPA) and found it to bind to the surface of OCs and, more importantly, to induce osteoclastogenesis.. In addition, the injection of MCV-ACPA into mice caused osteopenia (loss of bone mass) and increased osteoclastogenesis.. This was explained by an induction of TNF-α release from OC precursors and its consequent effect on the proliferation of these precursors and their expression of their activation makers (CD11b, CD14, CD115) and cytokine receptors (RANK).. Altogether, our.. in vitro.. and.. in vivo.. data provide evidence for a new pathological mechanism that links the adaptive immune response to bone remodelling: differentiating OCs increasingly express enzymes that induce protein (such as vimentin) citrullination.. ACPAs, in particular anti-MCV, are therefore released and target OC precursors, causing their self-activation and differentiation into OC.. This induced osteoclastogenesis is largely responsible for the increase of bone resorption and the decrease of bone mass in inflammatory conditions.. MCV-ACPAs induce bone loss in vivo.. (A) 3-dimensional (top) and 2-dimensional (bottom) μCT images of the tibial metaphysis of Rag1 / mice that were left untreated or treated with control IgG or MCV-ACPAs showing decreased bone volume, trabecular number, trabecular thickness and connectivity density.. (B) Microphotographs (original magnification, ×400) of tibial bones of Rag1 / mice treated with either IgG or MCV-ACPAs, stained for osteoclasts (purple stain and arrows) by histochemical detection of TRAP showing increased osteoclastogenesis following MCV-ACPA injection.. An analytical approach based on the concept of fluorophore localisation provides dynamic super-resolution data of xFP- labelled live cells using a common arc lamp based wide-field fluorescence microscope.. One method of achieving fluorescence super-resolution is based around finding the positions of fluorescent molecules that label the cellular structure of interest.. In this approach, positions can be determined precisely and accurately using fluorescent probes that can be photoactivated, photoconverted or photoswitched to generate single images with emitter densities of only about one active fluorophore per diffraction-limited area.. Many images each containing subsets of active fluorophores are collected and  ...   of mononucleated cells belonging to the monocyte/macrophage lineage.. They are found on the bone surface, where they remove the old bone matrix in order to allow bone rejuvenation by osteoblasts.. In order to adhere to their substrates, to migrate and to resorb, they are equipped of structures called podosomes, which are loosely organized on artificial substrates (plastic or glass) but condensed on calcium apatite mineral.. Podosomes are made of polymerized actin organized as dense dots, and are surrounded by a loose network of polymerized actin.. They are very dynamic structures with a short life-time (2 to 3 minutes), that self organize with different patterns during osteoclast life.. Podosomes are thought to be responsible for cell adhesion and migration.. In this article we show that osteoclast podosomes are major anchoring sites exerting tensions on the substrate.. They appear in areas where membranes are expanding and disappear in areas where membranes retract.. Videomicroscopy has shown that podosome dynamics can move latex beads dispersed in a soft substrate, thus demonstrating podosome-mediated tension forces.. Finally, analyzing the migration dynamic of several osteoclasts, we were able to show that actin-containing podosomes are major driving forces for osteoclast migration.. A mathematical model of osteoclast migration provides unexpected results: osteoclasts are moving by jumps and this is reminiscent of the way that osteoclasts are moving to resorb bone surfaces.. Dynamic images of an osteoclast spreading on the plastic surface of a petri dish.. Podosomes are in green; they are present at the periphery of the cell in the spreading area.. t= 0.. rounded non-adherent osteoclasts are seeded in the petri dish; Then images of the same cells 12minutes (t= 12 mn) and 20 minutes (t=20mn) after seeding.. Tumour growth and dissemination throughout the body to form metastases, the main cause of cancer-related mortality, depend on the ability of tumour cells to move through/invade the very dense meshwork formed by the extracellular matrix (ECM), in which the cells that make up tissues and organs are embedded.. The controlled degradation of specific components of the ECM is an essential step in tumour cell invasion.. This process can be recapitulated in the test tube using tumour cell lines that form protrusive structures called invadopodia that have the ability to locally degrade and penetrate the ECM.. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete.. In this paper, we describe a novel approach to accelerate the discovery process.. In detail, we were able to prepare tumour cell fragments enriched in invadopodia which were then analyzed by proteomic techniques.. This led to the discovery of new protein components belonging to different functional groups including those related to the production of energy, cell movement and secretion of proteins.. We expect these findings to open further avenues for the molecular study of invasive growth behaviour of cancer cells.. The G protein Beta subunit is a very important protein that mediates communication between the extracellular and intracellular environments.. We first discovered it to be associated with invadopodia in our proteomics analysis and then validated the finding by fluorescence microscopy as shown here.. Invadopodia (labeled in blue) are positioned over areas of digested extracellular matrix (dark holes in the green background) and contain the G protein Beta subunit (red).. Podosomes and invadopodia are dynamic, actin-rich adhesion structures and represent the two founding members of the invadosome family.. These plasma membrane microdomains are commonly found in cells of the immune system or in osteoclasts (podosomes) as well as in cancer cells (invadopodia), where they have been extensively described.. More recently, it was realized that other cells which do not spontaneously assemble invadosomes can be induced to do so under selective stimulation, such as a soluble factor, matrix receptor, cell stress or oncogenes.. Invadosomes harbour metalloproteases which degrade components of the extracellular matrix.. Because of this distinctive feature, invadosomes have been systematically linked with invasion processes but hints for other functions are now emerging.. During the last decade, tremendous advances have been made regarding the molecular mechanism underlying their formation, dynamics and function.. 3D analysis of invadosomes is now being actively developed in ex vivo and in vivo models to demonstrate their involvement in physiological and pathological processes.. Schematic representation of the evolution of models for invadosome analysis.. (C) In vitro invadosome observation in 3D and their presumed role are reported in a few publications.. (D) The same conclusion holds true for ex vivo or in vivo observation.. Macrophages are immune cells that form the first line of defense against infectious agents such as bacteria or viruses.. To get to sites of infection, macrophages have to migrate through the dense meshwork of the extracellular matrix that connects cells within tissues.. Podosomes, adhesion structures with the ability to locally degrade matrix material, play a key role in this migration.. We show here that matrix degradation by macrophage podosomes depends on contact with the microtubule network of cells.. Microtubules form a cellular railway system , which enables the delivery of cargo molecules through transport by tiny motors walking on these fibers.. This study identifies the motor protein KIF9 as an essential regulator of matrix degradation at podosomes.. KIF9 delivers cargo vesicles, which contain proteins of the reggie family, a group of membrane-associated proteins.. We could also identify a distinct region of KIF9 as the binding site for reggie-1.. Interaction of both proteins through this region is critical for regulating matrix degradation at podosomes.. Vesicles containing the motor protein KIF9 (green) are associated with microtubules (red), which form the cellular railway system.. Podoplanin is a small transmembrane glycoprotein whose expression is up-regulated in different types of cancer, especially in squamous cell carcinomas (SCCs).. Podoplanin up-regulation in tumour cells has been linked to enhanced cell motility and invasiveness; however, the mechanisms underlying this process remain poorly understood.. Since podoplanin lacks any obvious enzymatic motif within its structure, all these activities have to be mediated by protein protein interactions, highlighting the need to identify its binding partners.. To further shed light into the biology and function of this glycoprotein, we attempted to identify the key podoplanin binding partners involved in podoplanin-mediated cancer cell migration.. In this article, we show that CD44s, the standard isoform of the major hyaluronan (HA) receptor, is a novel partner for podoplanin during podoplanin-mediated migration.. We first show that the expression of both molecules is co-ordinately up-regulated in a mouse skin model of carcinogenesis during progression to highly aggressive cell carcinomas.. Second, we demonstrate that podoplanin interacts with CD44s both.. in vitro.. , by co-immunoprecipitation assays, and.. , by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM).. FRET/FLIM demonstrated that the association of these two proteins in the plasma membrane increases in cells with a migratory phenotype.. Importantly, we also show for the first time that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in squamous carcinoma cells.. Podoplanin CD44s complexes at the plasma membrane are up-regulated in cells with a migratory phenotype.. Multiphoton FLIM was used to image FRET between podoplanin-eGFP (donor) and CD44s-mRFP (acceptor) in MDCK cells.. This technique enables visualisation and quantification of protein protein interactions by analysis of the donor lifetime decay kinetics.. Interaction of podoplanin-eGFP and CD44s-mRFP was observed in single polarized cells, as measured by decreases in eGFP donor fluorescence lifetime (red) relative to its lifetime in control cells (blue) expressing podoplanin-eGFP alone (A).. FRET was localised at the trailing edge during rear retraction, and on small foci that were distributed throughout the apical surface of the cell across the lamellae (B).. Note that although co-localisation between podoplanin eGFP and CD44s mRFP was always detected, FRET was recorded mainly in isolated cells (i.. , those that had detached from their neighbours)..

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  • Title: Links
    Descriptive info: 7th Framework Programme of the European Commission:.. http://cordis.. europa.. eu/fp7/home_en.. html.. Initial Training Networks (ITNs):.. eu/fp7/mariecurieactions/itn_en.. European Commission Research Executive Agency (REA):.. http://ec.. eu/research/rea/index.. cfm?pg=home.. The Invadosome Consortium:.. invadosomes.. org.. The Cell Migration Consortium:.. cellmigration.. org/.. Job advertisement via EURAXESS:.. eu/euraxess/.. Marie Curie fellows association:.. mcfa.. eu/..

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