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. 2022 Mar;51(2):157-169.
doi: 10.1007/s00249-021-01576-8. Epub 2021 Oct 28.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

Steven Huth  1 , Johannes W Blumberg  2 , Dimitri Probst  2 , Jan Lammerding  3 , Ulrich S Schwarz  2 , Christine Selhuber-Unkel  4
Affiliations

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

Steven Huth et al. Eur Biophys J. 2022 Mar.

Abstract

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

Keywords: Cell adhesion; Mechanobiology; Micromanipulation; Traction force microscopy.

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Figures

Fig. 1
Fig. 1
A, B show exemplary phase contrast images of a microneedle shearing a PDMS pillar. The needle moves downwards and bends the pillar. The pillar, on the other hand, exerts a force to the needle, which results in a bending of the needle. The force acting between needle and pillar is calculated from the bending of the PDMS pillar. C presents a plot of the pillar force versus the bending of the microneedle for each frame of the shearing experiment. The slope corresponds to the microneedle’s spring constant
Fig. 2
Fig. 2
A microneedle is inserted into a fibroblast, which expresses fluorescently labeled zyxin and adheres to a TFM substrate. Subsequently, the needle is moved into the y-direction at a constant speed and exerts shear forces to the cell until it is detached. A, B Exemplary phase contrast images taken during cell shearing. Both the cell and the needle bending are monitored. The bending of the needle is used to calculate the shear force. C The cell’s zyxin distribution prior to the shearing process is recorded via fluorescence microscopy. D Traction force map of the cell with adhesion search areas delimited by white rectangles and mean patch locations marked by crosses. Traction forces were reconstructed for t = 0 s using Fourier Transform Traction Cytometry (FTTC). For the reconstruction of traction forces with the shear force monopole present (t > 0), we used the circular patch method. E The needle force and the cell’s net traction force are plotted as a function of time. F The traction forces in y-direction are plotted for different adhesion patches (labeled in panel D) to quantify how the cell loads its adhesion sites under the external shearing stimulus.
Fig. 3
Fig. 3
Comparison of the traction forces predicted in the absence of an external force monopole (at t = 0) using the circular patch method (that we employed during this study) and a regularized Fourier Transform Traction Cytometry (FTTC) (Schwarz and Soiné 2015) using generalized cross-validation (Huang 2019). A Profile for the cells introduced in Fig. 2. B Profile for the cells introduced in Fig. 5. In both cases, the agreement between the two methods is rather good
Fig. 4
Fig. 4
Change of force monopole and dipole moments of the cell presented in Fig. 2 in response to needle shearing. A presents the magnitudes of the force monopole, as well as the major and minor dipole moments and the torque as functions of time. Our results show that the contractile forces are initially distributed mostly isotropically around the contractile center. However, the force monopole created by the needle shearing increases over time while the minor dipole, which describes the contractility in the direction of the force, decreases only slightly. B shows the force monopole and the major dipole moment in exemplary force maps recorded during the shearing experiment. The force monopole is denoted by red arrows while the dipole moment is represented by purple arrows. The gray encircled regions represent areas where adhesions are predicted from the cell’s zyxin distribution
Fig. 5
Fig. 5
The change of traction forces as a response to microneedle shearing. A, B show phase contrast images of a cell adhering to a PAAm substrate and a microneedle exerting shear forces to the cell. The cell’s zyxin distribution prior to the shearing process is presented in C while D pictures a traction force map with cell’s adhesion patch positions marked with white crosses and numbers. The force map is calculated using FTTC at t = 0 s. In E The shear force exerted by the needle to the cell is compared to the magnitude of the net traction force vector. The y-components of the traction vectors for the adhesion patches are plotted for each moment of the experiment in F
Fig. 6
Fig. 6
Change of force monopole and dipole moments of the cell presented in Fig. 5 in response to needle shearing. A presents the magnitudes of the force monopole, as well as the major and minor dipole moments and the torque as functions of time. Our results show that the force balance is initially governed by the major dipole moment. However, the force monopole created by the needle shearing increases over time and governs the force balance at high shearing forces. B shows the force monopole and the major dipole moment in exemplary force maps recorded during the shearing experiment. The force monopole is denoted by red arrows while the dipole moment is represented by purple arrows. The gray encircled regions represent areas where adhesions are predicted from the cell’s zyxin distribution
Fig. 7
Fig. 7
Redistribution of adhesion patch loading after a rupture event. A, B show phase contrast images of a microneedle shearing a fibroblast on a PAAm substrate. The cell’s zyxin distribution is visualized in C. A map of the traction forces at t = 0 s exerted at the cell’s adhesion patches is presented in D. The white crosses mark the cell’s adhesion sites. The force map is calculated using FTTC at t = 0 s. The sum of traction forces has roughly the same magnitude as the external shear force, as can be seen in E. The y-components of the traction vectors for the adhesion patches are plotted in F. The dashed line marks the rupture of adhesion patches 1, 2 and 3 at t = 32 s
Fig. 8
Fig. 8
Change of force monopole and dipole moments of the cell presented in Fig. 7 in response to needle shearing. A Presents the magnitudes of the force monopole, as well as the major and minor dipole moments and the torque as functions of time. Our results show that the force balance is initially governed by the major dipole moment. However, the force monopole created by the needle shearing increases over time and governs the force balance at high shearing forces. B shows the force monopole and the major dipole moment in exemplary force maps recorded during the shearing experiment. The force monopole is denoted by red arrows while the dipole moment is represented by purple arrows. The gray encircled regions represent areas where adhesions are predicted from the cell’s zyxin distribution
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References

    1. Aramesh M, et al. Functionalized bead assay to measure three-dimensional traction forces during t-cell activation. Nano Lett. 2020;21:507–514. doi: 10.1021/acs.nanolett.0c03964. - DOI - PubMed
    1. Balaban NQ, et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol. 2001;3:466–472. doi: 10.1038/35074532. - DOI - PubMed
    1. Bischofs IB, Klein F, Lehnert D, Bastmeyer M, Schwarz US. Filamentous network mechanics and active contractility determine cell and tissue shape. Biophys J. 2008;95(7):3488–96. doi: 10.1529/biophysj.108.134296. - DOI - PMC - PubMed
    1. Brangwynne CP, et al. Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement. J Cell Biol. 2006;173:733–741. doi: 10.1083/jcb.200601060. - DOI - PMC - PubMed
    1. Brugués A, et al. Forces driving epithelial wound healing. Nat Phys. 2014;10:683–690. doi: 10.1038/NPHYS3040. - DOI - PMC - PubMed

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