Keywords: Innate immunity, ROS production, protein interaction, fluorescence microscopy Internship Duration: 30/11/-1 - 30/11/-1
Head of the hosting team: Oliver Nüsse
Website: Click here
Address of the host laboratory: Institute of Physical Chemistry Team Physical Chemistry of Biological Systems Bldg 350, Av Jean Perrin 91405 Orsay France
Supervisor 1: Oliver NüsseE-mail: oliver.nusse@universite-paris-saclay.fr Phone: +33 0169157644
Supervisor 2: Marie ErardE-mail: marie.erard@universite-paris-saclay.frPhone: +33 01 69 15 30
We are interested in the NAPDH oxidase of phagocytic cells. This membrane complex of 6 proteins is one of the most important sources of ROS (Reactive Oxygen Species), which are essential for the destruction of microbes by the mammalian immune system [1]. Insufficient ROS production leads to chronic infections whereas overproductionn leads to tissue damage in inflammatory diseases. Our aim is to identify the events that trigger the assembly of NADPH oxidase at the membrane and induce enzyme activation and ROS production. These steps are essential for identifying effective pharmacological modulators. To study assembly of the NADPH oxidase we use Forster-like resonant energy transfer (FRET) between the proteins of the complex. We use oxidase subunits tagged with fluorescent proteins that will give a FRET signal when they are assembled in a complex [2, 3]. In this project, FRET will be detected by Fluorescence Lifetime Imaging (FLIM) and a new method of flow cytometry (CytoFRET). We have phagocytic cells expressing the gp91 membrane subunit of NADPH oxidase fused to a fluorescent protein. The person recruited will characterize this line in terms of expression of the tagged subunit, its functionality and localization during phagocytosis. FRET-FLIM experiments will then be used to monitor assembly with cytosolic subunits. These experiments will reveal the spatio-temporal dynamics of NADPH oxidase assembly during phagocytosis. Since the FRET efficiency strongly depends on the distance between the two fluorescent proteins, il will also provide critical information on the spatial conformation of the complex. This internship is aimed at someone wishing to work in an interdisciplinary environment at the frontier between physical chemistry (fluorescence spectro-microscopy) and cell biology (phagocytic cells).
The intern will use numerous techniques from cell biology, molecular biology, biochemistry and physical chemistry. He/she will use cell culture and differentiation as well as transient transfection. He/she will use basic cloning techniques to create and amplify expression vectors. The cells will be caracterized by western blot, luminescence ROS assay, flow cytometry and fluorescence microscopy (confocal, spinning disk and FLIM). New data analysis tools developped in the laboratory will be used to extract quantitative information from flow cytometry and microscopy.
[1] Dupre-Crochet (2013) J Leuk Biol 94(4):657 doi: 10.1189/jlb.1012544 [2] Ziegler (2019) J Biol Chem 294(11):3824 doi:10.1074/jbc.RA118.006864 [3] Valenta (2022) BBA 1869:119276. doi: 10.1016/j.bbamcr.2022.119276
