High-resolution Traction Force Microscopy Research Articles

High-resolution traction force microscopy on small focal adhesions - improved accuracy through optimal marker distribution and optical flow tracking

The accurate determination of cellular forces using Traction Force Microscopy at increasingly small focal attachments to the extracellular environment presents an important yet substantial technical challenge. In these measurements, uncertainty regarding accuracy is prominent since experimental calibration frameworks at this size scale are fraught with errors – denying a gold standard against which accuracy of TFM methods can be judged. Therefore, we have developed a simulation platform for generating synthetic traction images that can be used as a benchmark to quantify the influence of critical experimental parameters and the associated errors. Using this approach, we show that TFM accuracy can be improved >35% compared to the standard approach by placing fluorescent beads as densely and closely as possible to the site of applied traction. Moreover, we use the platform to test tracking algorithms based on optical flow that measure deformation directly at the beads and show that these can dramatically outperform classical particle image velocimetry algorithms in terms of noise sensitivity and error. We then report how optimized experimental and numerical strategy can improve traction map accuracy, and further provide the best available benchmark to date for defining practical limits to TFM accuracy as a function of focal adhesion size.

Quantitative Analyses of Dynamic Features of Fibroblasts on Different Protein-Coated Compliant Substrates.

Cell response to substrate rigidity, closely related to extracellular matrix protein composition, requires actomyosin-generated contractility. By introducing coefficients describing cell spreading and traction dynamics, and a revised high-resolution traction force microscopy, we analyzed the static and dynamic features of fibroblasts on fibronectin- or collagen- coated stiff or soft substrates. Large cell spreading area and branchlike morphology were more favorable on fibronectin than collagen. Cell spreading on fibronectin-coated substrates was more sensitive to rigidity compared with collagen. Low concentration fibronectin-coated substrate induced more dynamic lamellipodia movement than other conditions. Interestingly, the static average cell traction on high concentration fibronectin-coated stiff and soft substrates showed no difference. However, the lamellipodium traction dynamics was sensitive to rigidity on fibronectin. Particularly, lamellipodia on fibronectin-coated soft substrate performed much higher local traction dynamics compared with other groups. Together, dynamics of cell adhesion and traction are regulated by extracellular matrix protein composition, coupled with substrate rigidity.

Correlative Traction Force Microscopy and Fluorescence Fluctuation Analysis of Molecular Aggregation and Complex Formation in Cell Adhesions in Distinct Microenvironments

The mechanical properties of the cellular microenvironment regulate processes that include migration, proliferation, and differentiation. These interactions occur at adhesions, which serve as both traction points and signaling hubs and mediate bi-directional sensing and responses to specific features of the surrounding extracellular matrix (ECM). Adhesions execute these activities through an intricate network of putative molecular interactions that largely remain to be demonstrated and characterized functionally in living cells. The challenge is to capture the highly localized and transient associations that characterize these activities in adhesions and determine how they respond to different microenvironments. In this study, we use high-resolution fluorescence fluctuation microscopy to map the formation and stoichiometry of integrin-associated complexes in the adhesions that populate the leading edge of migrating cells. We focus on putative integrin activating (kindlin and talin) and actin-linking (talin, vinculin and a-actinin) molecules and show that all molecules are present in adhesions as soon as they are visible; however, they form integrin containing complexes hierarchically, at different times, with variable stoichiometry within the adhesion itself and change as the adhesion matures into larger structures. To parse out the effects of the mechanical properties of the ECM on the numbers, aggregation states, and associations of these molecules, we extend these measurements to substrates with variable stiffness and correlate them with high-resolution traction force microscopy (TFM) measurements. We show that individual and newly formed adhesions at the leading edge of protruding cells transmit forces on soft as well as stiff substrates, with force magnitudes that correlate with the integrated intensity of the adhesions and the total number of individual adhesion molecules. These measurements provide novel information on complex formation as adhesions evolve and respond to substrate rigidity.

Feedback Interactions between Intracellular Contraction and Leading Edge Protrusion in Directed Cell Migration

Embryonic development depends on effective cell migration whose malfunction leads to abnormalities. Migration is the integrated outcome of a cycle of inter-connected component processes, namely protrusion, adhesion and contraction. Most, if not all, molecular details of these processes have been established. The major remaining challenge is to identify mechanisms that couple these processes in space and time. Compared to a relatively well-established interaction between protrusion and adhesion, however, there is no understanding as to how contraction and adhesion interact dynamically at the time scale of a single migration cycle, and whether these interactions affect protrusion through adhesion-protrusion coupling. The major hurdle that makes it challenging to investigate contraction- protrusion link is from technology: there is nearly no tool to quantify myosin II- based contraction in cytoskeletal network compared to numerous imaging approaches for characterization of protrusion - adhesion coupling. Here, we hypothesize that contraction dynamically modulates adhesion at a distance, which in turn promotes or inhibits protrusion via several redundant mechanical and signaling pathways. To test this hypothesis, we developed a continuum mechanical (CM) model to infer location and time of intracellular forces in migrating cells, which will be compared against high-resolution traction force microscopy (TFM) to obtain absolute force levels and infer material heterogeneity in the cytoskeleton. Preliminary results from Ptk1 cell wound-healing assay show that intracellular force field and traction force field are highly correlated, suggesting the feasibility of the absolute intracellular force level reconstruction. To establish the ‘information flow’ between contraction, adhesion and protrusion, we will use a correlation analysis of spontaneous fluctuations to show the coupling and information flow between them in unperturbed cells and in a cell where candidate molecules mediating the putative link between contraction and protrusion is slightly perturbed.

A Critical Pull to Polarize the Cell

A Critical Pull to Polarize the Cell

Chapter 20 - High-Resolution Traction Force Microscopy

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.

High Resolution Traction Force Microscopy Based on Experimental and Computational Advances

Cell adhesion and migration crucially depend on the transmission of actomyosin-generated forces through sites of focal adhesion to the extracellular matrix. Here we report experimental and computational advances in improving the resolution and reliability of traction force microscopy. First, we introduce the use of two differently colored nanobeads as fiducial markers in polyacrylamide gels and explain how the displacement field can be computationally extracted from the fluorescence data. Second, we present different improvements regarding standard methods for force reconstruction from the displacement field, which are the boundary element method, Fourier-transform traction cytometry, and traction reconstruction with point forces. Using extensive data simulation, we show that the spatial resolution of the boundary element method can be improved considerably by splitting the elastic field into near, intermediate, and far field. Fourier-transform traction cytometry requires considerably less computer time, but can achieve a comparable resolution only when combined with Wiener filtering or appropriate regularization schemes. Both methods tend to underestimate forces, especially at small adhesion sites. Traction reconstruction with point forces does not suffer from this limitation, but is only applicable with stationary and well-developed adhesion sites. Third, we combine these advances and for the first time reconstruct fibroblast traction with a spatial resolution of ∼1 μm.