Culture substrate stiffness impacts human myoblast contractility-dependent proliferation and nuclear envelope wrinkling.

Understanding how biophysical and biochemical microenvironmental cues together influence the regenerative activities of muscle stem cells and their progeny is crucial in strategizing remedies for pathological dysregulation of these cues in aging and disease. In this study, we investigated the cell-level influences of extracellular matrix (ECM) ligands and culture substrate stiffness on primary human myoblast contractility and proliferation within 16 h of plating and found that tethered fibronectin led to stronger stiffness-dependent responses compared to laminin and collagen. A proteome-wide analysis further uncovered cell metabolism, cytoskeletal and nuclear component regulation distinctions between cells cultured on soft and stiff substrates. Interestingly, we found that softer substrates increased the incidence of myoblasts with a wrinkled nucleus, and that the extent of wrinkling could predict Ki67 (also known as MKI67) expression. Nuclear wrinkling and Ki67 expression could be controlled by pharmacological manipulation of cellular contractility, offering a potential cellular mechanism. These results provide new insights into the regulation of human myoblast stiffness-dependent contractility response by ECM ligands and highlight a link between myoblast contractility and proliferation.

Alarming and Calming: Opposing Roles of S100A8/S100A9 Dimers and Tetramers on Monocytes.

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).

Human endothelial cells display a rapid tensional stress increase in response to tumor necrosis factor-α.

Endothelial cells form the inner layer of blood vessels, making them the first barrier between the blood and interstitial tissues; thus endothelial cells play a crucial role in inflammation. In the inflammatory response, one important element is the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). While other pro-inflammatory agents like thrombin and histamine induce acute but transient changes in endothelial cells, which have been well studied biologically as well as mechanically, TNF-α is primarily known for its sustained effects on permeability and leukocyte recruitment. These functions are associated with transcriptional changes that take place on the timescale of hours and days. Here, we investigated the early mechanical action of TNF-α and show that even just 4 min after TNF-α was added onto human umbilical vein endothelial cell monolayers, there was a striking rise in mechanical substrate traction force and internal monolayer tension. These traction forces act primarily at the boundary of the monolayer, as was to be expected. This increased internal monolayer tension may, in addition to TNF-α's other well-studied biochemical responses, provide a mechanical signal for the cells to prepare to recruit leukocytes.

Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

Pressure Drives Rapid Burst-Like Coordinated Cellular Motion from 3D Cancer Aggregates.

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL-1 ). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell-ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion.

Multi-oscillation microrheology via acoustic force spectroscopy enables frequency-dependent measurements on endothelial cells at high-throughput.

Active microrheology is one of the main methods to determine the mechanical properties of cells and tissue, and the modelling of these viscoelastic properties is under heavy debate with many competing approaches. Most experimental methods of active microrheology such as optical tweezers or atomic force microscopy based approaches rely on single cell measurements, and thus suffer from a low throughput. Here, we present a novel method for frequency-dependent microrheology on cells using acoustic forces which allows multiplexed measurements of several cells in parallel. Acoustic force spectroscopy (AFS) is used to generate multi-oscillatory forces in the range of pN-nN on particles attached to primary human umbilical vein endothelial cells (HUVEC) cultivated inside a microfluidic chip. While the AFS was introduced as a single-molecule technique to measure mechanochemical properties of biomolecules, we exploit the AFS to measure the dynamic viscoelastic properties of cells exposed to different conditions, such as flow shear stresses or drug injections. By controlling the force and measuring the position of the particle, the complex shear modulus G*(ω) can be measured continuously over several hours. The resulting power-law shear moduli are consistent with fractional viscoelastic models. In our experiments we confirm a decrease in shear modulus after perturbing the actin cytoskeleton via cytochalasin B. This effect was reversible after washing out the drug. Additionally, we include critical information for the usage of the new method AFS as a measurement tool showing its capabilities and limitations and we find that for performing viscoelastic measurements with the AFS, a thorough calibration and careful data analysis is crucial, for which we provide protocols and guidelines.

Energy level alignment at organic/inorganic semiconductor heterojunctions: Fermi level pinning at the molecular interlayer with a reduced energy gap.

The electronic properties of the organic/inorganic semiconductor heterojunction formed by para-sexiphenyl (6P) and three different faces of ZnO are investigated using photoelectron spectroscopy and X-ray absorption. While multilayer molecules stand almost upright with respect to the surface plane, we evidence the presence of a lying 6P interlayer, which exhibits a higher electron affinity. This is due to an energy gap narrowing because of the close vicinity of that interlayer to the higher dielectric constant ZnO and a more planar molecular conformation compared to molecules in the bulk. Both effects have a significant impact on the level alignment mechanisms at the three interfaces, i.e., surface electron push-back and Fermi level pinning. We disentangle the contribution of each effect to the level alignment for both standing and lying 6P layers and show that on ZnO(0001[combining macron]) only the push-back contributes, while on ZnO(101[combining macron]0) and ZnO(0001) Femi level pinning occurs in addition. In all three cases the lying 6P interlayer establishes the same work function to which the levels of the 6P multilayer align. Only the identification of the complex interplay of level alignment mechanisms and molecular degrees of freedom allows deriving a reliable picture of the energy levels at this heterojunction. This is important as the presence of an interlayer and its modified electronic states might go unnoticed, and conclusions on the correlation between purported interfacial energy levels and functionality of such semiconductor heterojunctions could be misleading.

Theory of mind in patients with schizophrenia: is mentalizing delayed?

OBJECTIVE: Functional imaging studies have used numerous neurocognitive designs to investigate brain activation during theory of mind (ToM) tasks in patients with schizophrenia. The majority of studies asks participants to retrospectively attribute mental states to others. We used a novel animated task to investigate implicit mentalizing online. Because behavioral studies have revealed slower ToM performance reaction times in patients with schizophrenia, we hypothesized that time would influence functional MRI (fMRI) activation patterns also. METHODS: We applied the "Moving Shapes" paradigm, which involves two interacting triangles, to a functional MRI block design and investigated the neural activation patterns of 15 patients with schizophrenia and 14 healthy controls. We additionally analyzed the first and second halves of each video separately to assess time-related differences. RESULTS: Overall, patients with schizophrenia showed increased activation in the inferior and middle frontal gyri, the superior temporal gyrus, the precuneus and the cerebellum compared with controls during ToM versus non-ToM videos. Most importantly, patients with schizophrenia had predominantly increased activation in ToM-related brain areas during the second half of the ToM paradigm, whereas the activation in areas of the ToM-network in healthy controls occurred during the first half of the presentation. CONCLUSIONS: Our results confirm recent findings of significantly stronger neural activations that encompass the fronto-temporo-parietal cerebral areas in patients with schizophrenia compared with controls during ToM tasks. The observation of slower cognitive processing in patients with schizophrenia during mentalizing might explain some of the contradictory imaging findings in these patients and have implications for cognitive remediation.