Dosimetry of an in vitro Exposure System for Fluorescence Measurements at 2.45 GHz

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Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 A resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis

The Kinetics of Cell Adhesion to Solid Scaffolds: An Experimental and Theoretical Approach

The process of cell seeding on biocompatible scaffolds has a major impact on the morphological evolution of an engineered tissue because it involves all the key factors of tissue formation: cells, matrix, and their mutual interactions. In order to characterize the efficiency of cell seeding techniques, mainly static parameters are used such as cell density, cell distribution, and cell viability. Here, we present an experimental model that incorporates an optical density meter providing real-time information on the cell seeding velocity, a relevant dynamic parameter of cell–matrix interaction. Our setup may be adapted to fit various cell seeding protocols. A modified fluorimetric cuvette is used as bioreactor culture flask. The optical density of the magnetically stirred cell suspension is recorded by a digital optoelectronic device. We performed calibration experiments in order to prove that, in our experimental conditions, optical density depends linearly on the number of cells in the unit volume of suspension. Control studies showed that, during the time course of a typical experiment (up to 10 h), the cells (murine 3T3 fibroblasts) neither aggregated nor adhered significantly to the walls of the cuvette. Hence, our setup yields the number of cells attached to the scaffold as a function of time. In order to analyze the experimental seeding curves, we built a kinetic model based on Langmuir’s adsorption theory, which was extended to include a preliminary step of integrin function recovery. We illustrate the proposed approach by two sets of experiments that involved trypsin–EDTA or only EDTA treatment (no trypsin) used to detach the cells from the culture flasks. The data indicate that in both cases cell–matrix adhesion has a sequential, two-step dynamics, but kinetic parameters and attachment site availability depend on the experimental protocol