Quantifying Traction Stresses in Adherent Cells

Kraning-Rush, Casey M.; Carey, Shawn P.; Califano, Joseph P.; Reinhart‐King, Cynthia A.

doi:10.1016/b978-0-12-388403-9.00006-0

Cited by 63 publications

(70 citation statements)

References 104 publications

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“…The volumes described herein will create a gel that has a thickness of ≈30 µm on 22 × 40 mm coverslips. Since the extent of polymer swelling varies with the PAAG formulation (Kraning-Rush et al, 2012) and cannot be easily predicted based on shear modulus alone, it is important to measure the height of the resulting TFM substrate. The gel must be sufficiently thick such that the gel can freely deform due to cellular forces without the influence of the underlying glass (Sen, Engler, & Discher, 2009).…”

Section: 1 Materialsmentioning

confidence: 99%

“…By applying known forces, Harris et al were able to calibrate this technique and to assess the magnitude of traction forces. However, limitations of this approach include difficulty in force quantification due to the nonlinearity of the silicone rubber deformation and low spatial resolution (Beningo & Wang, 2002; Kraning-Rush, Carey, Califano, & Reinhart-King, 2012). Further development of this approach, which combined high-resolution optical imaging and extensive computational procedures, dramatically improved the resolution, accuracy, and reproducibility of traction force measurements and transformed TFM into a technique with relatively wide use in many biomedical research laboratories (Aratyn-Schaus & Gardel, 2010; Dembo & Wang, 1999; Gardel et al, 2008; Lee, Leonard, Oliver, Ishihara, & Jacobson, 1994; Ng, Besser, Danuser, & Brugge, 2012).…”

Section: Introductionmentioning

confidence: 99%

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High-Resolution Traction Force Microscopy

Plotnikov

Sabass

Schwarz

et al. 2014

Methods in Cell Biology

201

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Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research.

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Section: 1 Materialsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High-Resolution Traction Force Microscopy

Plotnikov

Sabass

Schwarz

et al. 2014

Methods in Cell Biology

201

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“…Cellular mechanosensing consists in probing the environment and influences cell decisions regarding functions such as migration, differentiation and morphogenesis. Numerous studies have focused on understanding how focal adhesions (FAs) exert traction forces onto the substratum using a variety of techniques known as traction force microscopy, where markers of substrate deformation (such as embedded microbeads, microprinted patterns or flexible micropillarsy) 1 are tracked and their displacements converted into forces. Podosomes are another adhesion structures suspected to play a role in mechanosensing, as podosome-related structures, invadopodia from breast cancer cells and podosome rosettes formed in genetically modified fibroblasts have been shown to sense the environment stiffness [2][3][4][5][6][7] .…”

mentioning

confidence: 99%

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes

et al. 2014

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Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

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“…It is clear that ECM stiffness is directly linked with the extent of tension generated at focal adhesion sites; therefore, by measuring cell traction forces, TFM may probe the mechanical properties of the local ECM microenvironment. [81][82][83] In a TFM experiment, cells are cultured on an optically clear polyacrylamide substrate coated with ECM constituents, and the motion of fluorescent fiduciary microbeads embedded within the gel is tracked. Traction forces are then determined by analyzing bead displacements during cell migration via the digital cross-correlation of images obtained during adhesion (stressed state) and following detachment (unstressed state) (Fig.…”

Section: Traction Force Microscopymentioning

confidence: 99%

Optical measurement of arterial mechanical properties: from atherosclerotic plaque initiation to rupture

Nadkarni

2013

J. Biomed. Opt

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Abstract. During the pathogenesis of coronary atherosclerosis, from lesion initiation to rupture, arterial mechanical properties are altered by a number of cellular, molecular, and hemodynamic processes. There is growing recognition that mechanical factors may actively drive vascular cell signaling and regulate atherosclerosis disease progression. In advanced plaques, the mechanical properties of the atheroma influence stress distributions in the fibrous cap and mediate plaque rupture resulting in acute coronary events. This review paper explores current optical technologies that provide information on the mechanical properties of arterial tissue to advance our understanding of the mechanical factors involved in atherosclerosis development leading to plaque rupture. The optical approaches discussed include optical microrheology and traction force microscopy that probe the mechanical behavior of single cell and extracellular matrix components, and intravascular imaging modalities including laser speckle rheology, optical coherence elastography, and polarization-sensitive optical coherence tomography to measure the mechanical properties of advanced coronary lesions. Given the wealth of information that these techniques can provide, optical imaging modalities are poised to play an increasingly significant role in elucidating the mechanical aspects of coronary atherosclerosis in the future.

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Quantifying Traction Stresses in Adherent Cells

Cited by 63 publications

References 104 publications

High-Resolution Traction Force Microscopy

High-Resolution Traction Force Microscopy

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes

Optical measurement of arterial mechanical properties: from atherosclerotic plaque initiation to rupture

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