Modulation of leukocyte adhesiveness is critical to leukocyte function during the immune response. In order to extravasate from the blood stream, leukocyte rolling must be followed by integrin-mediated rapid arrest. Intergins play a crucial role on chemokine-induced arrest of leukocytes on blood vessels. Signaling events mediating adhesion are extensively studied. Increasing evidence underlays the essential role of lipid second messenger as important fine regulators of signaling cascade leading to integrin affinity modulation. To date, no technology has ever been developed to monitor intracellular production and localization of specific lipids in the context of leukocyte recruitment. Imaging of small molecules in real time in living cells is usually accomplished with genetically encoded sensors, which are typically fluorescent proteins flanking a ligand-binding domain. However, sensor development is difficult since proteins undergoing conformational changes upon binding a desired target molecule are minimally available. In this scenario, my project aims to produce sensors for fluorescence imaging of small molecules using RNA. These RNA-based sensors comprise a ligand-binding RNA aptamer and Spinach, an aptamer that binds and switches on the fluorescence of a small- molecule fluorophore allowing imaging of the dynamic changes and cell-to-cell variation in the intracellular levels of phosphatidic acid (PA) and phosphatidil-inositol-4,5-biphospate (PIP4,5P2). This tool could be the first of this kind and could be useful to study many aspects of signaling cascade events, not only for leukocyte recruitments. By using lipid Nano-Biosensor I could be able to make FRET and FRAP studies in order to obtain in vivo qualitative and quantitative data allowing topological and dynamic reconstruction of signaling networks. Moreover lipid Nano-Biosensors could be developed as innovative new markers for cell sorting in FACS and in ImageStream Technology.
Get Access to the 1st Network for European Cooperation