Human adipocyte function is impacted by mechanical cues.

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity-induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri-adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL-6, G-CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro-inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri-adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT-dependent CTGF promoter activity, which depends on ß1-integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co-activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT-induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation.

Traction forces exerted by epithelial cell sheets.

Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

Homophilic interactions between cadherin fragments at the single molecule level: an AFM study.

We report measurements of the adhesion forces between single E-cadherin fragments anchored on solid surfaces. These fragments consist of the two outermost extracellular domains of the protein. The specificity of the measured rupture forces was demonstrated by Ca2+ exchange experiments. Two series of experiments were performed using two linkers of different rigidity and length. We find that the pull-off force is distributed with a maximum value independent of the linker and logarithmically dependent on the velocity of separation of the two surfaces. Our dynamical results are compatible with previous flow chamber experiments performed with the same fragments and can be compared from a different perspective with previously reported AFM experiments on the full-length extracellular domain of the VE-cadherin. Interestingly, using a rigid linker, we have been able for the first time to evidence the deformation of the cadherin molecule under mechanical stress, a piece of information not accessible with more classical grafting strategies.

Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.

Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained.