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

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function

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

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

Membrane tension controls the phase equilibrium in fusogenic liposomes

Fusogenic liposomes have been widely used for molecule delivery to cell membranes and cell interior. However, their physicochemical state is still little understood. We tested mechanical material behavior by micropipette aspiration of giant vesicles from fusogenic lipid mixtures and found that the membranes of these vesicles are fluid and under high mechanical tension even before aspiration. Based on this result, we developed a theoretical framework to determine the area expansion modulus and membrane tension of such pre-tensed vesicles from aspiration experiments. Surprisingly high membrane tension of 2.1 mN m−1 and very low area expansion modulus of 63 mN m−1 were found. We interpret these peculiar material properties as the result of a mechanically driven phase transition between the usual lamellar phase and an, as of now, not finally determined three dimensional phase of the lipid mixture. The free enthalpy of transition between these phases is very low, i.e. on the order of the thermal energy

Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusio

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM–ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM–ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini