Mechanical characterization of cell signaling mechanisms.

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The two aims of this research program are designed to explore the relationship between G-proteins coupled receptors (GPCR) signalling and mechanical properties and function in vascular cells. 1) RELATION BETWEEN CELL MEMBRANE TENSION AND GPCR MECHANICAL ACTIVATION. Transduction of mechanical force into biochemical information trough the activation of cell signaling pathways plays a central role in physiological and pathophysiological processes. Cell membrane proteins acting as mechanosensor includes a variety of ion channels, phospholipases and integrins. More recently, a growing body of experimental evidence suggested that G-proteins coupled receptors (GPCR) could be implicated as mechanosensor, in particular the AT1 receptor of angiotensin II, which has received a significant attention. Indeed, the AT1 receptor (AT1-R) can be activated by mechanical stimuli in absence of agonist, a peculiar functional property believed to be involved in cardiovascular function. The mechanisms responsible for GPCR activation by mechanical forces are still poorly understood. Two models are currently debated as to how GPCR transduce force. The first model involves its interaction with macromolecular elements present at the cell surface of within the cell cytoskeleton, which effectively act as a mechanical antenna to perceive the external mechanical perturbation. The second model supposes that GPCR are activated due to a perturbation of the cell membrane lateral tension, which affect the pressure profile at the GPCR/phospholipid bilayer interface. Such changes in membrane lateral tension provoke conformational changes of GPCR and shift their activation status. In this objective, we propose original AFM experiments to investigate the role of cell membrane tension in GPCR activation. 2) IMPACT OF SELECTIVE ANGIOTENSIN SIGNALLING ON VASCULAR SMOOTH MUSCLE CELLS MECHANICAL PHENOTYPE. An important property of the vascular smooth muscle cells (VSMC) is the plasticity required for the function, adaptation and maintenance of the cardiovascular system. In normal physiological conditions, the contractile phenotype of functional VSMC is regulated through the expression of genes encoding contractile and cytoskeletal proteins, intracellular enzymes, as well as cell surface ligands and receptors. However, in response to vascular stress, VSMCs down-regulate the expression of contractile proteins and activates the cell cycle, which leads to the so-called “synthetic” phenotype. VSMC phenotype switching is the hallmark of vascular function function regulation. Myocardin (Myocd) was recently demonstrated as a key player in the regulation of the contractile phenotype of VSMC. Myocd interacts with the MADS box transcription factor, SRF, to upregulate the transcription of genes encoding for a number of proteins of the actomyosin contractile machinery such as Myosin Heavy Chain (MyHC) and SM-a-actin. In this situation, the Myocd transactivation domain can be phosphorylated by Erk 1/2 leading to a decrease of the VSMC contraction marker. NFkB was also demonstrated to interact with Myocd to inhibit its activity, which may explain phenotypic switch observed in VSMC in inflammatory conditions. Therefore, it is expected that perturbations of the Erk 1/2 and NFkB signaling pathways by canonical (AngII) and biased (SII) ligands of AT1-R could differently affect the contractile phenotype of VSMC. In this objective, using VSMC models, we will study the link existing between Erk 1/2, NFkB signaling, the modulation of contractile markers, the mechanical properties of VSMC (isometric tension) as well as the contractile capability of individual cells.