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Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation

View ORCID ProfileMorteza Aramesh, Simon Mergenthal, Marcel Issler, View ORCID ProfileBirgit Plochberger, Florian Weber, View ORCID ProfileXiao-Hua Qin, View ORCID ProfileRobert Liska, View ORCID ProfileGeorg N. Duda, View ORCID ProfileJohannes B. Huppa, View ORCID ProfileJonas Ries, View ORCID ProfileGerhard J. Schütz, View ORCID ProfileEnrico Klotzsch
doi: https://doi.org/10.1101/2020.09.23.310144
Morteza Aramesh
1Laboratory of Applied Mechanobiology, Department for Health Sciences and Technology, ETH Zürich, 8093 Zürich, Switzerland
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Simon Mergenthal
2Institute of Biology, Experimental Biophysics/ Mechanobiology, Humboldt Universität zu Berlin, 10115 Berlin, Germany
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Marcel Issler
2Institute of Biology, Experimental Biophysics/ Mechanobiology, Humboldt Universität zu Berlin, 10115 Berlin, Germany
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Birgit Plochberger
3Upper Austria University of Applied Sciences, Campus Linz, Garnisonstrasse 21, 4020 Linz, Austria
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Florian Weber
3Upper Austria University of Applied Sciences, Campus Linz, Garnisonstrasse 21, 4020 Linz, Austria
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Xiao-Hua Qin
4Institute for Biomechanics, Department for Health Sciences and Technology, ETH Zürich, 8093 Zürich, Switzerland
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Robert Liska
5Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163/MC, 1060, Vienna, Austria
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Georg N. Duda
6Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration, Charité-Universitätsmedizin Berlin
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Johannes B. Huppa
7Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
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Jonas Ries
8EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
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Gerhard J. Schütz
9Institute of Applied Physics, TU Wien, Vienna, Austria
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  • For correspondence: schuetz@iap.tuwien.ac.at enrico.klotzsch@hu-berlin.de
Enrico Klotzsch
1Laboratory of Applied Mechanobiology, Department for Health Sciences and Technology, ETH Zürich, 8093 Zürich, Switzerland
2Institute of Biology, Experimental Biophysics/ Mechanobiology, Humboldt Universität zu Berlin, 10115 Berlin, Germany
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  • For correspondence: schuetz@iap.tuwien.ac.at enrico.klotzsch@hu-berlin.de
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Abstract

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts.

Significance statement During the antigen recognition process, T-cells explore and probe their environment via microvilli, which exert local pushes and pulls at the surface of the antigen presenting cell. It is currently believed that these forces influence or even enable the antigen recognition process. Here, we describe the development of a platform, which allows us to quantify the magnitude and direction of traction forces exerted locally by T cell microvilli. Simultaneous Ca2+ imaging was used to link the measured forces to the overall T cell activation status. Superresolution microscopy resolved the contact sites of bead-microvilli contact at the nanoscale: cells contacted beads via actin vortex-like structures, which excluded the phosphatase CD45 from the contacts.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 24, 2020.
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Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation
Morteza Aramesh, Simon Mergenthal, Marcel Issler, Birgit Plochberger, Florian Weber, Xiao-Hua Qin, Robert Liska, Georg N. Duda, Johannes B. Huppa, Jonas Ries, Gerhard J. Schütz, Enrico Klotzsch
bioRxiv 2020.09.23.310144; doi: https://doi.org/10.1101/2020.09.23.310144
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Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation
Morteza Aramesh, Simon Mergenthal, Marcel Issler, Birgit Plochberger, Florian Weber, Xiao-Hua Qin, Robert Liska, Georg N. Duda, Johannes B. Huppa, Jonas Ries, Gerhard J. Schütz, Enrico Klotzsch
bioRxiv 2020.09.23.310144; doi: https://doi.org/10.1101/2020.09.23.310144

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