Dynamic monitoring of cell mechanical properties using profile microindentation.

We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells' actin is depolymerized using cytochalasin-D.

Tensile forces govern germ-layer organization in zebrafish.

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

Membrane tensiometer for heavy giant vesicles.

One key parameter of giant-vesicles adhesion is their membrane tension, sigma. A theoretically simple but delicate way to impose (and measure) it is to use micropipette manipulation techniques. But usually, the vesicles are free and their tension is unknown, until an adhesion patch grows. Sigma can be deduced from the detailed profile of the membrane close to the substrate, but this method is limited to very low tensions. We present here a rather simple way to estimate the membrane tension of heavy vesicles, which sediment close to a surface, by observing by RIM the size of the flat region of the vesicle. As an application, we follow the slow flattening of vesicles, when the surrounding sugar solution is evaporating, and their light-induced tensioning.

Adhesion between giant vesicles and supported bilayers decorated with chelated E-cadherin fragments.

Here, we present a study of adhesion between cadherin fragments using giant unilamellar vesicles and supported bilayers. These objects are partially made of nickel chelating lipids and are subsequently decorated with proteins bearing a 6His tag. Initially, we observed their fixation and correct orientation by using a fluorescent protein, the green fluorescent protein (GFP)-6His. The adhesive behavior of E-cadherin functionalized giant vesicles and supported bilayers was studied as a function of the calcium concentration and of the protein functionality by reflection interference microscopy. We show that such a system retains specific cadherin-mediated adhesion and could be used to study the statics and dynamics of adhesive plaques as well as to gain insight into the fundamental mechanisms of cellular adhesion at the mesoscopic scale.

Line thermodynamics: adsorption at a membrane edge.

We report a novel experimental study of line thermodynamics. Our system consists of detergent molecules adsorbing at the edges of freestanding lipid bilayers. Adsorption reduces the line tension T of the membrane edges. Measuring T as a function of the bulk detergent concentration C, we obtain a line adsorption isotherm. Using an extension of Gibbs's surface thermodynamics to lines, we estimate the "line excess density" of adsorbants and the energy of adsorption per site.