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Seminario DFBMC-IFIBYNE – Expone: Dr. Piotr Bregestovski “Using of genetically encoded biosensor for non-invasive analysis of ion concentrations and ion channels activation”

iCal Import
Octubre 19, 2018
Libre y gratuito
13 horas, Aula de Seminarios del LFBM
11 4576 3368
Pabellón II, 2º piso., Ciudad Universitaria, CABA, Argentina

Piotr Bregestovski (invitado especial)
Aix-Marseille University, INSERM, INS, Institut de Neurosciences des Systèmes, 13005 Marseille, France.
e-mail: pbreges@gmail.com

Genetically encoded probes have become powerful tools for non-invasive imaging of concentrations of ions, the activity of enzymes, distribution of small molecules, proteins and organelles, and the protein interactions in living cells. Discovery that the native fluorescence of yellow fluorescent protein (YFP) is quenched by anions led to the development of genetically encoded intracellular halide sensors. We developed several molecular probes for non-invasive fluorescent monitoring of intracellular Cl ([Cl]i) and the functioning of Cl-selective ionotropic receptors for glycine (GlyR) and GABA. These biosensors are used either at the transient expression in cultured cells or organisms or at stable expression producing transgenic animals possessing heritable and functional probes. Using the mouse Thy1 mini-promoter, we generated two lines of transgenic mice for the monitoring of intracellular chloride (Cl−) and for the simultaneous measurements of intracellular Cl−and pH. The first line expresses a CFP-YFP-based Cl probe called Cl-Sensor and exhibited a good biosensor expression in neurons of the hippocampus and cortex. The second line expresses ClopHensor, which consists of a pH and chloride sensitive variant of enhanced green fluorescent protein (E2GFP) fused with red fluorescent protein (DsRed). To reveal the ClopHensor expression pattern across the brain of transgenic mice we obtained transparent brain samples using the advanced CLARITY method and imaged them with confocal and light-sheet microscopy. This analysis also provides the map of the brain areas biosensors. These types of probes offer a means of screening pharmacological agents and monitoring Cl and pH under different physiological and pathological conditions.

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