Measuring cellular traction forces on non-planar substrates

Animal cells use traction forces to sense the mechanics and geometry of their environment. Measuring these traction forces requires a workflow combining cell experiments, image processing and force reconstruction based on elasticity theory. Such procedures have been established before mainly for planar substrates, in which case one can use the Green's function formalism. Here we introduce a worksflow to measure traction forces of cardiac myofibroblasts on non-planar elastic substrates. Soft elastic substrates with a wave-like topology were micromolded from polydimethylsiloxane (PDMS) and fluorescent marker beads were distributed homogeneously in the substrate. Using feature vector based tracking of these marker beads, we first constructed a hexahedral mesh for the substrate. We then solved the direct elastic boundary volume problem on this mesh using the finite element method (FEM). Using data simulations, we show that the traction forces can be reconstructed from the substrate deformations by solving the corresponding inverse problem with a L1-norm for the residue and a L2-norm for 0th order Tikhonov regularization. Applying this procedure to the experimental data, we find that cardiac myofibroblast cells tend to align both their shapes and their forces with the long axis of the deformable wavy substrate.Comment: 34 pages, 9 figure

Traction force microscopy with optimized regularization and automated Bayesian parameter selection for comparing cells

Adherent cells exert traction forces on to their environment, which allows them to migrate, to maintain tissue integrity, and to form complex multicellular structures. This traction can be measured in a perturbation-free manner with traction force microscopy (TFM). In TFM, traction is usually calculated via the solution of a linear system, which is complicated by undersampled input data, acquisition noise, and large condition numbers for some methods. Therefore, standard TFM algorithms either employ data filtering or regularization. However, these approaches require a manual selection of filter- or regularization parameters and consequently exhibit a substantial degree of subjectiveness. This shortcoming is particularly serious when cells in different conditions are to be compared because optimal noise suppression needs to be adapted for every situation, which invariably results in systematic errors. Here, we systematically test the performance of new methods from computer vision and Bayesian inference for solving the inverse problem in TFM. We compare two classical schemes, L1- and L2-regularization, with three previously untested schemes, namely Elastic Net regularization, Proximal Gradient Lasso, and Proximal Gradient Elastic Net. Overall, we find that Elastic Net regularization, which combines L1 and L2 regularization, outperforms all other methods with regard to accuracy of traction reconstruction. Next, we develop two methods, Bayesian L2 regularization and Advanced Bayesian L2 regularization, for automatic, optimal L2 regularization. Using artificial data and experimental data, we show that these methods enable robust reconstruction of traction without requiring a difficult selection of regularization parameters specifically for each data set. Thus, Bayesian methods can mitigate the considerable uncertainty inherent in comparing cellular traction forces

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration

The constant beat: cardiomyocytes adapt their forces by equal contraction upon environmental stiffening

Cardiomyocytes are responsible for the permanent blood flow by coordinated heart contractions. This vital function is accomplished over a long period of time with almost the same performance, although heart properties, as its elasticity, change drastically upon aging or as a result of diseases like myocardial infarction. In this paper we have analyzed late rat embryonic heart muscle cells' morphology, sarcomere/costamere formation and force generation patterns on substrates of various elasticities ranging from ∼1 to 500 kPa, which covers physiological and pathological heart stiffnesses. Furthermore, adhesion behaviour, as well as single myofibril/sarcomere contraction patterns, was characterized with high spatial resolution in the range of physiological stiffnesses (15 kPa to 90 kPa). Here, sarcomere units generate an almost stable contraction of ∼4%. On stiffened substrates the contraction amplitude remains stable, which in turn leads to increased force levels allowing cells to adapt almost instantaneously to changing environmental stiffness. Furthermore, our data strongly indicate specific adhesion to flat substrates via both costameric and focal adhesions. The general appearance of the contractile and adhesion apparatus remains almost unaffected by substrate stiffness.ISSN:2046-639

Changing the Way of Entrance: Highly Efficient Transfer of mRNA and siRNA via Fusogenic Nano-Carriers

Transferring nucleic acids into mammalian cells heavily influences life science for decades. While first applications mainly dealt with DNA transfer for various purposes as e.g., plasmid encoded protein expression or generation of mutant strains, subsequent applications additionally transferred RNA molecules of mainly small lengths for specific knockdown (RNAi) or site-specific genome modification (gRNA). Significant improvements in full length mRNA generation and extension of mRNA lifetimes additionally allows their use for transient expression in latest times. For all of these types of nucleic acids the most common cell incorporation method is based on complexation and subsequent endosomal uptake. This so-called lipofection can be used theoretically for almost any mammalian cell type and a tremendous number of different product compositions exist in order to deal with drawbacks as transfer efficiency, cell type selectivity, endosomal degradation, slow uptake and cytotoxicity. In contrast, new methods transfer complexed RNA molecules directly into the cytoplasm using liposomal nano-carriers that fuse with cellular plasma membranes immediately upon contact to free functional nucleic acids directly into the cytoplasm. Here, we compare both methods in detail with special focus on robustness, short- and long-term cytotoxicity, efficiency and functionality for various types of transferred RNA. Our data clearly indicate that direct RNA incorporation via fusogenic nano-carriers circumvents most endosomal uptake-based challenges, making it to a most promising alternative for nucleic acid transfer

Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms

BackgroundThe electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood.MethodsHere, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems.ResultsCell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+.ConclusionsThese findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts