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Charakterisierung der molekularen Mechanismen der a5ß1 and avß3 Integrin abhängigen Regulierung der Adhäsion von Fibroblasten

English title Characterizing the molecular mechanisms of a5ß1 and avß3 integrin crosstalk and catch bond of fibroblast adhesion
Applicant Müller Daniel Jobst
Number 160225
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 Biophysics
Start/End 01.11.2015 - 31.10.2018
Approved amount 673'000.00
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All Disciplines (3)

Discipline
Biophysics
Cellular Biology, Cytology
Molecular Biology

Keywords (9)

Single-cell force spectroscopy; Fibronectin; Fibroblasts; Shear stress enhanced adhesion; Receptor Crosstalk; Cell adhesion molecules; RGD binding site; Integrins; Cell adhesion

Lay Summary (German)

Lead
Menschliche und tierische Zellen nutzen Adhäsionsmoleküle (CAMs), um mit ihrer Umgebung zu wechselwirken. Dabei regulieren CAMs adhäsive Wechselwirkungen zelluläre Prozesse und Signalwege, die wiederum andere CAMs steuern. Diese Regulation wird von Zellen benötigt, um an ihre Umgebung zu adaptieren. Obwohl ein Großteil der in Adhäsionsmechanismen involvierten Proteine bekannt ist, sind die Mechanismen, über welche Zellen ihre Adhäsion regulieren, weitgehend unverstanden. Das Forschungsprojekt leistet einen Beitrag zu diesem Verständniss.
Lay summary

In diesem Projekt untersuchen wir die Regulationsmechanismen von Integrinen. Integrine bilden eine wichtige Familie innerhalb der CAMs tierischer und menschlicher Zellen und etablieren deren Adhäsion zur extrazellulären Matrix. Sobald Integrine einen extrazellulären Liganden binden, initiieren sie biochemische Signalwege der Zelle. Diese Signalwege können den Zellzyklus, -form, oder -mobilität beeinflussen. Durch die Aktivierung bestimmter Integrine durch Liganden können auch CAMs zur Zellmembran hinzugefügt werden oder in bestimmte funktionelle Zustände versetzt werden. Solche Mechanismen ermöglichen es Zellen schnell und flexibel auf die spezifische Erkennung ihrer Umgebung zu reagieren. Obwohl α5β1 und αvβ3 Integrine auf verschiedenen Wegen zur Regulation des Zytoskeletts beitragen können ist nicht bekannt wie beide Integrine kooperieren um die Zelladhäsion zu regulieren. Um diese Wirkungsweise beider Integrine zu untersuchen setzen wir moderne Kraftspektroskopische und Mikroskopische Methoden ein. Damit erhoffen wir uns wichtige Einsichten wie beide Integrine die Zelladhäsion funktionell und strukturell regulieren, wie sie miteinander Kommunizieren und zu wichtigen Funktionen betreffend des Aufbaus und Erhalts des extrazellulären Gewebes beitragen

Direct link to Lay Summary Last update: 18.09.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
Oscillatory Switches of Dorso-Ventral Polarity in Cells Confined between Two Surfaces
Helenius Jonne, Ecke Mary, Müller Daniel J., Gerisch Günther (2018), Oscillatory Switches of Dorso-Ventral Polarity in Cells Confined between Two Surfaces, in Biophysical Journal, 115(1), 150-162.
Virus stamping for targeted single-cell infection in vitro and in vivo
Schubert Rajib, Trenholm Stuart, Balint Kamill, Kosche Georg, Cowan Cameron S, Mohr Manuel A, Munz Martin, Martinez-Martin David, Fläschner Gotthold, Newton Richard, Krol Jacek, Scherf Brigitte Gross, Yonehara Keisuke, Wertz Adrian, Ponti Aaron, Ghanem Alexander, Hillier Daniel, Conzelmann Karl-Klaus, Müller Daniel J, Roska Botond (2017), Virus stamping for targeted single-cell infection in vitro and in vivo, in Nature Biotechnology, 36(1), 81-88.
Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding
Toyoda Yusuke, Cattin Cedric J., Stewart Martin P., Poser Ina, Theis Mirko, Kurzchalia Teymuras V., Buchholz Frank, Hyman Anthony A., Müller Daniel J. (2017), Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding, in Nature Communications, 8(1), 1266-1266.
Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second
Strohmeyer Nico, Bharadwaj Mitasha, Costell Mercedes, Fässler Reinhard, Müller Daniel J. (2017), Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second, in Nature Materials, 16(12), 1262-1270.
Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces
Newton Richard, Delguste Martin, Koehler Melanie, Dumitru Andra C, Laskowski Pawel R, Müller Daniel J, Alsteens David (2017), Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces, in Nature Protocols, 12(11), 2275-2292.
High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox
Laskowski Pawel R., Pfreundschuh Moritz, Stauffer Mirko, Ucurum Zöhre, Fotiadis Dimitrios, Müller Daniel J. (2017), High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox, in ACS Nano, 11(8), 8292-8301.
Imaging modes of atomic force microscopy for application in molecular and cell biology
Dufrêne Yves F., Ando Toshio, Garcia Ricardo, Alsteens David, Martinez-Martin David, Engel Andreas, Gerber Christoph, Müller Daniel J. (2017), Imaging modes of atomic force microscopy for application in molecular and cell biology, in Nature Nanotechnology, 12(4), 295-307.
Atomic force microscopy-based characterization and design of biointerfaces
Alsteens David, Gaub Hermann E., Newton Richard, Pfreundschuh Moritz, Gerber Christoph, Müller Daniel J. (2017), Atomic force microscopy-based characterization and design of biointerfaces, in Nature Reviews Materials, 2(5), 17008-17008.
Multiparametric Atomic Force Microscopy Imaging of Biomolecular and Cellular Systems
Alsteens David, Müller Daniel J., Dufrêne Yves F. (2017), Multiparametric Atomic Force Microscopy Imaging of Biomolecular and Cellular Systems, in Accounts of Chemical Research, 50(4), 924-931.
αV-class integrins exert dual roles on α5β1 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. (2017), αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin, in Nature Communications, 8, 14348-14348.
The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes
Benito-Jardón Maria, Klapproth Sarah, Gimeno-LLuch Irene, Petzold Tobias, Bharadwaj Mitasha, Müller Daniel J, Zuchtriegel Gabriele, Reichel Christoph A, Costell Mercedes (2017), The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes, in eLife, 6, e22264.
Nanomechanical mapping of first binding steps of a virus to animal cells
Alsteens David, Newton Richard, Schubert Rajib, Martinez-Martin David, Delguste Martin, Roska Botond, Müller Daniel J. (2016), Nanomechanical mapping of first binding steps of a virus to animal cells, in Nature Nanotechnology, 12(2), 177-183.
Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin
Theodosiou Marina, Widmaier Moritz, Böttcher Ralph T, Rognoni Emanuel, Veelders Maik, Bharadwaj Mitasha, Lambacher Armin, Austen Katharina, Müller Daniel J, Zent Roy, Fässler Reinhard (2016), Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin, in eLife, 5, e10130.

Collaboration

Group / person Country
Types of collaboration
Prof. Reihard Fässler, Max Planck Institute of Biochemistry, Department of Molecular Medicine Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Mercedes Costell, University Valencia Spain (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Dr. Botond Roska, Friedrich Miescher Institut Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
SNI Annual Meeting 2018 Talk given at a conference Mechanically Detecting and Directing Molecular and Cellular Processes towards Controlling Organisms 13.09.2018 Lenzerheide, Switzerland Müller Daniel Jobst;
The Future of Nanoscience Talk given at a conference Mechanically Detecting and Directing Molecular and Cellular Processes towards Controlling Organisms 04.09.2018 Tutzing, Germany Müller Daniel Jobst;
New Horizon in Membrane Transport and Communication Talk given at a conference Atomic Force Microscopy-based Mechanobiology 16.08.2018 Frankfurt, Germany Müller Daniel Jobst;
2nd Biointerfaces International Talk given at a conference Measuring the mass regulation of single mammalian cells at millisecond time resolution 15.08.2018 Zürich, Switzerland Müller Daniel Jobst;
Gordon Research Conference: Signaling by Adhesion Receptors - Multi-Scale Adhesion Mechanics and Signaling Poster Quantification of Cell Adhesion Modulation during Mitosis 24.07.2018 Maine, United States of America Huber Maximilian; Antunes dos Santos Marilia;
Dutch Biophysics Meeting Talk given at a conference Studying Mechanical Processes of Life from the Cellular to Molecular Scale 02.10.2017 Veldhoven, Netherlands Müller Daniel Jobst;
Building a synthetic cell Talk given at a conference Engineering Molecular Factories and Cellular Systems towards Health Control 12.07.2017 Ringberg, Germany Müller Daniel Jobst;
FEBS Workshop – Biological Surfaces and Interfaces: Interface Dynamics Poster Upon binding bronectin α5β1 integrins sense mechanical load and within a second signal to reinforce adhesion 02.07.2017 Sant Feliu de Guixols, Spain Strohmeyer Nico;
Colloquium on the Physics of Living Systems Talk given at a conference Imaging biological structure and functioning, with a focus on atomic force microscopy 02.04.2017 Brüssel, Belgium Müller Daniel Jobst;
Annual Meeting of the German Biophysical Society (DGfB) Talk given at a conference Molecular Mechanics Guiding Cell Shape Changes in Mitosis 25.09.2016 Erlangen, Germany Müller Daniel Jobst;
Biomembrane Days Talk given at a conference Cut-and-paste of single integral membrane proteins 05.09.2016 Berlin, Germany Müller Daniel Jobst;
Swiss Nanoconvention Talk given at a conference Atomic Force Microscopy to Study Processes of Life from the Cellular to Molecular Scale 30.06.2016 Basel, Switzerland Müller Daniel Jobst;
Gordon research Conference (GRC) on Signaling by Adhesion Receptors Poster 5ß1 integrins employ catch bonds to activate fibronectin-binding integrins via FAK and c-Src to reinforce fibroblast adhesion 18.06.2016 Lewiston ME, United States of America Strohmeyer Nico;
Gordon Research Conference (GRC) on Biointerface Science Talk given at a conference Single Molecule Atomic Force Microscopy and Force Spectroscopy for Quantifying Biological Processes 12.06.2016 Diablerets, Switzerland Müller Daniel Jobst;
3rd NovAliX Conference - Biophysics in Drug Discovery, Talk given at a conference Imaging of G-protein coupled receptors while quantifying their ligand-binding free energy landscape to mutiple ligands 07.06.2016 Strasbourg, France Müller Daniel Jobst;


Self-organised

Title Date Place

Associated projects

Number Title Start Funding scheme
182587 Characterizing the cell cycle dependent regulation of adhesion to extracellular matrix proteins 01.05.2019 Project funding (Div. I-III)
138063 Systematische Charakterisierung der Kommunikation von Zelladhäsionsrezeptoren mittels Einzelzellkraftspektroskopie 01.09.2012 Project funding (Div. I-III)
138063 Systematische Charakterisierung der Kommunikation von Zelladhäsionsrezeptoren mittels Einzelzellkraftspektroskopie 01.09.2012 Project funding (Div. I-III)

Abstract

Integrins comprise a diverse family of cell adhesion molecules (CAMs) that establish cell adhesion and have key roles in the assembly of cells into functional tissues and organs. The interactions between integrins are believed to be responsible for two major functions of cell adhesion sites: first, their role as a ‘scaffold’ in supporting the physical integration of the cell to the extracellular matrix (ECM) and the cytoskeleton, which leads to the assembly of tissues with a particular morphological and mechanical properties; second, their ‘signaling’ activity, which enables cells to sense the chemical and mechanical properties of the external environment and to respond by activating signaling networks that regulate cell structure, dynamics, behavior and fate. Mammalian cells usually co-express several integrins, which recognize ECM components by binding short and specific amino acid stretches such as the Arg-Gly-Asp (RGD) motif. From the family of integrins, a5ß1 and all av-class integrins recognize and bind the RGD motif, which is found in a multitude of ECM proteins including fibronectin. However, although a5ß1 and avß3 integrins have distinct functions in regulating the actin cytoskeleton, the cooperation of both integrins in facilitating cell adhesion to ECM proteins exposing the RGD motif is not yet understood. In collaboration with Dr. Reinhard Fässler (MPI, Martinsried), we started to discern the distinct roles of two fibronectin binding integrins, avß3 and a5ß1 in fibroblast adhesion. Therefore, Fässler provided genetically engineered mouse kidney fibroblasts that express avß3 and/or a5ß1 integrins as fibronectin binding cell adhesion molecules. We found that integrin- fibronectin interaction in early fibroblast adhesion (=2 min) depends strongly on the specific set of integrins expressed. In fibroblasts expressing both heterodimers, cell adhesion was lower than for cells expressing only a5ß1 implicating a negative regulation. Thus, we observed the cross-regulation of a5ß1 and avß3 integrins. Surprisingly, we found that the activation and the subsequent signaling of avß3 integrins increase the adhesion of fibroblasts to fibronectin via a5ß1 integrins. This indicates a clear crosstalk between a5ß1 and avß3 integrins. Furthermore, it has been shown that the functional synergy between a5ß1 and av-class integrin signaling hubs is required to establish focal-adhesion-mediated rigidity sensing which play an avid role in mechanotransduction and migratory processes. Yet, it remains unexplained how a5ß1 and avß3 integrins are involved in regulating fibroblast adhesion. We will thus utilize single-cell force spectroscopy (SCFS) in combination with modern optical microscopy (e.g. TIRF, fluorescence, confocal) and atomic force microscopy (AFM)-based methods to quantitatively investigate pertinent aspects of cell adhesion, including integrin crosstalk, shear dependent adhesion, integrin localization and catch bond behavior of integrins. Therefore, we will closely work with Fässler, who will provide us numerous genetically engineered fibroblasts (e.g. knock-outs of av, ß1, ILK, talin, kindlin).In the proposed funding period we aim to achieve the following goals:i) Unravel integrin induced crosstalk that signals enhanced a5ß1 adhesionii) Characterize crosstalk dependent shear enhanced adhesion of single integrins and single cells iii) Delineate integrin localization and ligand-binding using high-resolution functional AFM imagingThis comprehensive study will provide valuable information on how a5ß1 and avß3 integrins regulate cell adhesion to fibronectin and hence delineate their distinct (and co-operative) role in an adhesome. It will also throw light on localization pattern of integrins, which governs the biophysical nature of their interaction with the ECM and dynamic cytoskeleton. The understanding of these mechanisms will not only appreciate normal physiological processes such as development and wound healing but also aid the therapeutics for progression of various inflammatory diseases and cancer.
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