We introduce a novel three-dimensional (3D) traction force microscopy (TFM) method motivated by the recent discovery that cells adhering on plane surfaces exert both in-plane and out-of-plane traction stresses. We measure the 3D deformation of the substratum on a thin layer near its surface, and input this information into an exact analytical solution of the elastic equilibrium equation. These operations are performed in the Fourier domain with high computational efficiency, allowing to obtain the 3D traction stresses from raw microscopy images virtually in real time. We also characterize the error of previous two-dimensional (2D) TFM methods that neglect the out-of-plane component of the traction stresses. This analysis reveals that, under certain combinations of experimental parameters (cell size, substratums' thickness and Poisson's ratio), the accuracy of 2D TFM methods is minimally affected by neglecting the out-of-plane component of the traction stresses. Finally, we consider the cell's mechanosensing of substratum thickness by 3D traction stresses, finding that, when cells adhere on thin substrata, their out-of-plane traction stresses can reach four times deeper into the substratum than their in-plane traction stresses. It is also found that the substratum stiffness sensed by applying out-of-plane traction stresses may be up to 10 times larger than the stiffness sensed by applying in-plane traction stresses.

The extracellular matrix (ECM) is a complex, dynamic network present within all tissues and organs that not only acts as a mechanical support and anchorage point but can also direct fundamental cell behavior, function, and characteristics. Although the importance of the ECM is well established, the integration of well‐controlled ECMs into Organ‐on‐Chip (OoC) platforms remains challenging and the methods to modulate and assess ECM properties on OoCs remain underdeveloped. In this review, current state‐of‐the‐art design and assessment of in vitro ECM environments is discussed with a focus on their integration into OoCs. Among other things, synthetic and natural hydrogels, as well as polydimethylsiloxane (PDMS) used as substrates, coatings, or cell culture membranes are reviewed in terms of their ability to mimic the native ECM and their accessibility for characterization. The intricate interplay among materials, OoC architecture, and ECM characterization is critically discussed as it significantly complicates the design of ECM‐related studies, comparability between works, and reproducibility that can be achieved across research laboratories. Improving the biomimetic nature of OoCs by integrating properly considered ECMs would contribute to their further adoption as replacements for animal models, and precisely tailored ECM properties would promote the use of OoCs in mechanobiology.

Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell’s mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior–posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA− and abp120− cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.

Basic human functions such as breathing and digestion require mechanical stretching of cells and tissues. However, when it comes to laboratory experiments, the mechanical stretching that cells experience in the body is not often replicated, limiting the biomimetic nature of the studies and the relevance of results. Herein, we establish the importance of mechanical stretching during in vitro investigations by reviewing seminal works performed using cell-stretching platforms, highlighting important outcomes of these works as well as the engineering characteristics of the platforms used. Emphasis is placed on the compatibility of cell-stretching devices (CSDs) with live-cell imaging as well as their limitations and on the research advancements that could arise from live-cell imaging performed during cell stretching.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca²⁺-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection. Infections during pregnancy can lead to infections of the placenta, spread to the fetus, and cause fetal damage and death. Improving maternal-child heath is a global heath priority. Yet, progress to prevent and treat pregnancy-related diseases has lagged behind other medical fields. Using pregnant guinea pigs, which have a placental structure that closely resembles humans, we identified a protein (InlP) secreted by the bacterial pathogen Listeria monocytogenes that strongly promotes placental infection. In human placental organ cultures bacteria deficient in InlP were impaired in their ability to spread from infected placental cytotrophoblasts into the underlying fetal stroma. Here, we solved the crystal structure of InlP, and identified Afadin, a cytoplasmic protein that localizes to adherens junctions as a binding partner of InlP. We demonstrate that InlP decreases the magnitude of traction stresses epithelial cells exert on an underlying extracellular matrix, and furthermore, that InlP facilitates bacterial spread from infected epithelial monolayers into an underlying compartment. Our study provides new insights into the mechanisms of bacterial spread across the placental barrier.