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For more than a couple of decades now, “force” has been recognized as an important physical parameter that cells employ to adapt to their microenvironment. Whether it is externally applied, or internally generated, cells use force to modulate their various actions, from adhesion and migration to differentiation and immune function. T lymphocytes use such mechano-sensitivity to decipher signals when recognizing cognate antigens presented on the surface of antigen presenting cells (APCs), a critical process in the adaptive immune response. As such, many techniques have been developed and used to measure the forces felt/exerted by these small, solitary and extremely reactive cells to decipher their influence on diverse T cell functions, primarily activation. Here, we focus on traction force microscopy (TFM), in which a deformable substrate, coated with the appropriate molecules, acts as a force sensor on the cellular scale. This technique has recently become a center of interest for many groups in the “ImmunoBiophysics” community and, as a consequence, has been subjected to refinements for its application to immune cells. Here, we present an overview of TFM, the precautions and pitfalls, and the most recent developments in the context of T cell immunology [ABSTRACT FROM AUTHOR]

For more than a couple of decades now, “force” has been recognized as an important physical parameter that cells employ to adapt to their microenvironment. Whether it is externally applied, or internally generated, cells use force to modulate their various actions, from adhesion and migration to differentiation and immune function. T lymphocytes use such mechano-sensitivity to decipher signals when recognizing cognate antigens presented on the surface of antigen presenting cells (APCs), a critical process in the adaptive immune response. As such, many techniques have been developed and used to measure the forces felt/exerted by these small, solitary and extremely reactive cells to decipher their influence on diverse T cell functions, primarily activation. Here, we focus on traction force microscopy (TFM), in which a deformable substrate, coated with the appropriate molecules, acts as a force sensor on the cellular scale. This technique has recently become a center of interest for many groups in the “ImmunoBiophysics” community and, as a consequence, has been subjected to refinements for its application to immune cells. Here, we present an overview of TFM, the precautions and pitfalls, and the most recent developments in the context of T cell immunology.

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms. [ABSTRACT FROM AUTHOR]

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue. [ABSTRACT FROM AUTHOR]

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis., Competing Interests: Authors DS and RC were employed by Aluda Pharmaceuticals, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kim, Warrington, López-Guajardo, Al Hennawi, Cook, Griffith, Symmes, Zhang, Qu, Xu, Chen and Gad.)

Abstract: Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. [Copyright &y& Elsevier]

Accurate measurement of cellular traction force is critical for understanding physical interaction between cells and the extracellular matrix. Traction force microscopy (TFM) has become the most widely used tool for this purpose. While TFM has made continual progress in terms of resolution and accuracy, there have been challenges regarding obtaining user-friendly software and choosing the right values for parameters and sub-processes associated with the software. Here we provide step-by-step instructions for a MATLAB-based TFM software application equipped with multiple methods for image deformation quantification and force reconstruction, along with clarification on the computational meaning of the parameters within the software. We outline how to choose the optimal sub-methods and values for parameters for each process, depending on the characteristics of images and purpose of the analyses. The software's runtime is 20, 4, and 0.05 min by Fast BEM L1 (Boundary Element Method L1-regularization), Fast BEM L2 (L2-regularization), and FTTC (Fourier Transform Traction Cytometry), respectively, in addition to 7 min of particle-tracking velocimetry-based deformation tracking, for a single image (1280 × 960 pixel) on a standard workstation. Finally, the colocalization accuracies, in reference to a paxillin-GFP image, are compared between the three force reconstruction methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Setting up the TFM package in MATLAB Basic Protocol 2: Running the TFM package Alternate Protocol 1: Stage drift correction: Efficient subpixel registration Alternate Protocol 2: Force field calculation: FastBEM., (© 2021 Wiley Periodicals LLC.)

The ability for biological cells to produce mechanical forces is important for the development, function, and homeostasis of tissue. The measurement of cellular forces is not a straightforward task because individual cells are microscopic in size and the forces they produce are at the nanonewton scale. Consequently, studies in cell mechanics rely on advanced biomaterials or flexible structures that permit one to infer these forces by the deformation they impart on the material or structure. Herein, the scientific progression on the use of deformable materials and deformable structures to measure cellular forces are reviewed. The findings and insights made possible with these approaches in the field of cell mechanics are summarized., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)