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Systematische Charakterisierung der Kommunikation von Zelladhäsionsrezeptoren mittels Einzelzellkraftspektroskopie

English title Systematic examination of adhesion-receptor crosstalk using single-cell force spectroscopy
Applicant Müller Daniel Jobst
Number 138063
Funding scheme Project funding (Div. I-III)
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Cellular Biology, Cytology
Start/End 01.09.2012 - 31.10.2015
Approved amount 673'000.00
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All Disciplines (2)

Discipline
Cellular Biology, Cytology
Biophysics

Keywords (5)

Zelladhäsion; Crosstalk; Kraftspektroskopie; Zellbiologie; Zelladhäsionsrezeptoren

Lay Summary (German)

Lead
Lay summary

Systematische Charakterisierung der Kommunikation von Zelladhäsionsrezeptoren mittels Einzelzellkraftmikroskopie

 

Antragsteller: Prof. Daniel J. Müller, Departement für Biosysteme, ETH Zürich

Damit Zellen mit Substraten, Zellen oder Gewebe wechselwirken können, sind spezifische und steuerbare Adhäsionsmechanismen erforderlich. Obwohl ein Großteil der in Adhäsionsmechanismen involvierten Proteine bekannt ist, sind die Mechanismen, über die Zellen Adhäsion regulieren, unbekannt. Der Grund dafür liegt darin, dass konventionelle Methoden es kaum ermöglichen, quantitative und systemspezifische Informationen über Mechanismen, welche die Zelladhäsion regulieren, zu erzielen. Vor kurzem wurde die Einzelzellkraftspektroskopie (SCFS) eingeführt, welche es ermöglicht, die Mechanismen der Zelladhäsion bis hin zum Beitrag einzelner Moleküle zu quantifizieren. Ein Ziel dieses Antrages ist, die SCFS weiter zu entwickeln, so dass damit Bindungsspektren einzelner Zellen quantitativ und systematisch erfasst werden können.

Zellen exprimieren unterschiedliche Adhäsionsmoleküle (CAMs), um zu kontrollieren, wie stark und über welchen Zeitraum sie mit ihrer extrazellulären Umgebung adhärieren. Solche adhäsiven Wechselwirkungen regulieren auch zelluläre Prozesse und Signalwege, die wiederum andere CAMs steuern. Diese Regulation (engl. ‚Crosstalk’) zwischen CAMs wird von Zellen benötigt, um an ihre Umgebung zu adaptieren. Um die unterschiedlichen Crosstalks einer Zelle zu erfassen, werden wir SCFS weiter entwickeln, so dass die unterschiedlichen Beiträge verschiedener CAMs zur Zelladhäsion quantifiziert werden können. Um darüberhinaus die Signalwege und Entscheidungsprozesse zellulärer Crosstalks verstehen zu können, kombinieren wir SCFS mit modernen zellbiologischen und mikroskopischen Methoden. Vorläufige Resultate zeigen, dass es solche Versuchsanordnungen ermöglichen, zu beobachten, wie durch die spezifische Bindung eines bestimmten CAMs andere CAMs der Zelle aktiviert oder deaktiviert werden. Mit fortschreitender Entwicklung dieses auf SCFS basierenden Ansatzes, beabsichtigen wir in unterschiedlichen zellulären Systemen detaillierte intrazelluläre Regulationsmechanismen zwischen Adhäsionsrezeptoren systematisch und quantitativ zu erfassen.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
The biomechanical properties of an epithelial tissue determine the location of its vasculature
Kragl Martin, Schbert Rajib, Karsjens Haiko, Otter Silke, Bartosinska Barbara, Jeruschke Hay, Weiss Jürgen, Chen Chunguang, Alsteens David, Kuss Oliver, Speier Stephan, Eberhard Daniel, Müller Daniel J., Lammert Eckhard (2016), The biomechanical properties of an epithelial tissue determine the location of its vasculature, in Nature Communications, 7, 13560.
In PC3 prostate cancer cells ephrin receptors crosstalk to β1-integrins to strengthen adhesion to collagen type I
Yu Miao, Wang Jinghe, Muller Daniel J., Helenius Jonne (2015), In PC3 prostate cancer cells ephrin receptors crosstalk to β1-integrins to strengthen adhesion to collagen type I, in Scientific Reports, 5, 8206.
Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces
Miao Yu, Strohmeyer Nico, Wang Jinghe, Muller Daniel J., Helenius Jonne (2015), Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces, in Beilstein J. Nanotechnol, 6, 157-166.
Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement
Sorce Barbara, Escobedo Carlos, Toyoda Yusuke, Stewart Martin P., Cattin Cedric J., Newton Richard, Banerjee Indranil, Stettler Alexander, Roska Botond, Eaton Suzanne, Hyman Anthony A., Hierlemann Andreas, Müller Daniel J. (2015), Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement, in Nature Communications, 6, 8872.
Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6.
Te Riet Joost, Helenius Jonne, Strohmeyer Nico, Cambi Alessandra, Figdor Carl G, Müller Daniel J (2014), Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6., in Journal of cell science, 127(Pt 7), 1595-1606.
A practical guide to quantify cell adhesion using single-cell force spectroscopy.
Friedrichs Jens, Legate Kyle R, Schubert Rajib, Bharadwaj Mitasha, Werner Carsten, Müller Daniel J, Benoit Martin (2013), A practical guide to quantify cell adhesion using single-cell force spectroscopy., in Methods (San Diego, Calif.), 60(2), 169-178.
Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion, and neurite outgrowth using atomic force microscopy-based single-cell force spectroscopy.
Beckmann Jan, Schubert Rajib, Chiquet-Ehrismann Ruth, Müller Daniel J (2013), Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion, and neurite outgrowth using atomic force microscopy-based single-cell force spectroscopy., in Nano letters, 13(6), 2937-2946.
Quantifying cellular adhesion to covalently immobilized extracellular matrix proteins by single-cell force spectroscopy.
Friedrichs Jens, Werner Carsten, Müller Daniel J (2013), Quantifying cellular adhesion to covalently immobilized extracellular matrix proteins by single-cell force spectroscopy., in Methods in molecular biology (Clifton, N.J.), 1046, 19-37.
αV-integrins are required for mechanotransduction in MDCK epithelial cells.
Teräväinen Terhi P, Myllymäki Satu M, Friedrichs Jens, Strohmeyer Nico, Moyano Jose V, Wu Chuanyue, Matlin Karl S, Muller Daniel J, Manninen Aki (2013), αV-integrins are required for mechanotransduction in MDCK epithelial cells., in PloS one, 8(8), 71485-71485.
Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy.
Schubert Rajib, Strohmeyer Nico, Bharadwaj Mitasha, Ramanathan Subramanian P, Krieg Michael, Friedrichs Jens, Franz Clemens M, Muller Daniel J, Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy., in FEBS letters.
aV-class integrins exert dual roles on a5b1 integrins to strengthen adhesion to fibronectin
Bharadwaj Mitasha, Strohmeyer Nico, Colo Georgina P., Helenius Jonne, Beerenwinkel Niko, Schiller Herbert B., Fässler Reinhard, Müller Daniel J., aV-class integrins exert dual roles on a5b1 integrins to strengthen adhesion to fibronectin, in Nature Communications, 8, 14348.

Collaboration

Group / person Country
Types of collaboration
Reinhard Fässler, Max-Planck-Institute of Biochemistry, Martinsried, Germany Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Carsten Werner/Leibniz Institute of Polymer Research, Dresden Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Aki Manninen/Oulu University Finland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Ruth Chiquet-Ehrismann/Friedrich-Miescher-Institute Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Anthony Hyman/Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Karl Matlin/Chicago University United States of America (North America)
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
GRC on Fibronectin, Integrins & Related Molecules Poster Integrins under stress: How fibroblasts tune α5β1 and αVβ3 activity in response to mechanical stress 10.05.2015 Lucca, Italy Bharadwaj Mitasha;
GRC on Fibronectin, Integrins & Related Molecules Poster Systematic Examination Of Integrin α5β1 and αVβ3 Crosstalk 10.05.2015 Lucca, Italy Strohmeyer Nico; Bharadwaj Mitasha;
Gordon Research Conference (GRC) on Single Molecule Approaches to Biology Talk given at a conference Folding- and unfolding- pathways of transmembrane β-barrel proteins 13.07.2014 Lucca, Italy Müller Daniel Jobst;
International meeting of the German Society for Cell Biology Individual talk Deciphering Molecular Mechanisms Guiding Cell Shape in Mitotisis 18.03.2014 Regensburg, Germany Müller Daniel Jobst;
Bi-Annual Meeting of the Spanish Society for Chemical Biology Individual talk Deciphering Molecular Mechanisms Guiding Cell Shape in Mitotisis 04.02.2014 Bilbao, Spain Müller Daniel Jobst;
UK SPM 2013 Meeting Individual talk Deciphering Molecular Mechanics Guiding Cellular Processes by Atomic Force Microscopy 26.06.2013 Leeds, Great Britain and Northern Ireland Müller Daniel Jobst;
ELMI (European Light Microscopy Initiative) Individual talk Towards quantifying cellular mechanics: Tracking mitotic cells by combined light and atomic force microscopy 21.05.2013 Bordeaux, France Müller Daniel Jobst;


Self-organised

Title Date Place
European Biophysics Congress EBSA 18.07.2015 Dresden, Germany

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
BASAR MOLEKULAR Talk 22.10.2015 Basel, Switzerland Müller Daniel Jobst;


Communication with the public

Communication Title Media Place Year
Media relations: radio, television BASAR MOLEKULAR German-speaking Switzerland 2015

Associated projects

Number Title Start Funding scheme
134521 Multifunktionelles hochauflösendes Abbilden und Kartieren der Kraftfelder biologischer Membranen und Membranproteine 01.03.2012 Project funding (Div. I-III)
160225 Charakterisierung der molekularen Mechanismen der a5ß1 and avß3 Integrin abhängigen Regulierung der Adhäsion von Fibroblasten 01.11.2015 Project funding (Div. I-III)
160225 Charakterisierung der molekularen Mechanismen der a5ß1 and avß3 Integrin abhängigen Regulierung der Adhäsion von Fibroblasten 01.11.2015 Project funding (Div. I-III)

Abstract

Cells use specific and controllable adhesion mechanisms to interact with substrates, cells and tissue. Although most of the receptors involved in these adhesion mechanisms are known, the mechanisms by which they regulate adhesion remain largely unknown. The primary reason for this is that conventional cell adhesion assays are not suited to provide quantitative insights on mechanisms that regulate cell adhesion. Single-cell force spectroscopy (SCFS) allows the quantitative characterization of cellular adhesion mechanisms down to the contribution of single cell adhesion molecules (CAMs). We intend to expand and systematically implement SCFS to capture the broad mechanisms regulating the CAMs of living cells. Cells express and regulate CAMs to control whether or not, how strong and how long they adhere to surfaces. CAMs are receptors that form adhesive interactions, activate signal transduction pathways and regulate cellular processes. In regulatory processes termed ‘crosstalk’, CAMs regulate the activity of other CAMs. This allows the cell to actively adapt its adhesion properties to the environment. We have recently introduced SCFS to characterize such crosstalk in HeLa cells (Friedrichs et al., 2010b). For the first time, we observed that and quantified to which extend collagen type I-binding to a1ß1 integrin regulates cell adhesion mediated by a5ß1 integrin to fibronectin. Perturbation experiments showed that this crosstalk is unidirectional from a1ß1 to a5ß1 integrin and functioned by endocytosis of a5ß1 integrin. So far no systematic study or screen has been developed to identify which receptors crosstalk with CAMs and to quantify the extent this crosstalk modulates the adhesion of CAMs. Therefore, we will implement SCFS to systematically screen for crosstalk regulating adhesion of various CAMs in different human and vertebrate cells. In addition to crosstalk between CAMs, the implemented SCFS assay will allow us to determine how extracellular signals such as growth factors regulate adhesive properties of cells. The SCFS-based screening will focus on proteins known to induce cellular responses. Therefore, we will collaborate with several research groups that have a broad expertise on different extracellular matrix (ECM) proteins and CAMs, and in the molecular cell biology and genetics of the cellular systems. The collaborators will provide us with various substrates, cells and genetic tools (i.e. RNAi constructs, GFP-tagged receptors and CAMs, molecular compounds, specific blocking antibodies) to manipulate cellular system and CAMs. This unique combination of cellular systems, and of molecular cell biological and genetic tools will be essential to examine the mechanisms of the crosstalk networks discovered. Furthermore, the SCFS-based experiments will be performed in combination with advanced optical microscopy (i.e. fluorescence, confocal, TIRF, structured illumination, spinning disc) to provide structural insights into cellular mechanisms regulating crosstalk. This unique combination of nano- and microscopic tools will allow us to study to which extent the dynamic reassembly of the actomyosin cortex or of cell surface receptors modulates the adhesion of a given CAM. While our SCFS-based screens will focus on a wide array of cell surface receptors and cellular systems, it will provide a general unbiased array of ECM proteins that induce crosstalk with CAMs and of intercellular signaling proteins that are involved in this crosstalk. This project will not only be the first systematic quantitative characterization of the adhesive state of cells and its regulation by extracellular factors, but include follow up examination of specific mechanisms of the crosstalks discovered.
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