Biochemistry and Cell Biology
Tuesday 6 April 2010
Dr. M. Wauben en dr. R. Wubbolts
Design of a model system to study exosome trafficking based on selective tetraspanin incorporation.
Exosomes are membranous nanoparticles that are secreted from a wide variety of cells. Although described for over two decades, only recently exosomes have been recognized as a new means of communication between cells (Reviewed in Thery 2009). Exosomes are composed of a selected set of (membrane) proteins and RNA species derived from the secreting cell and thus provide selective combinatorial signals from one cell to another. The molecular packing of exosomes relies on endosomal sorting mechanisms which determine the ultimate exosomal composition. We study the role of exosome formation and function in immune cells. This project focuses on the development of a model system that will allow visualization of exosome formation in dendritic cells and subsequent traffic of exosomes towards T cells.
Tetraspanin molecules are a family of multimembrane spanning glycoproteins that perform a wide variety of functions in cells. They form microdomains within membranes by homotypic and heterotypic interactions. Inclusion of adhesion and signaling molecules within these networks leads to the shaping functional subdomains within membranes (Reviewed in Hemler 2005).
The tetraspanins, CD9, CD37, CD63 and CD81 have been found in dendritic cell exosomes and may serve to selectively collect molecules that can be included into these exosomes. However, the relative contribution of these different tetraspanins remains unclear. We aim to characterize exosomal incorporation of these tetraspanin by expressing their mGFP tagged versions in dendritic cells and determine relative enrichment in exosomes when compared to cellular levels. Regulation of exosome production and release from dendritic cells by T cell contacts will be studied in parallel. The mGFP tag allows fluorescence microscopy to determine intracellular sorting of the tetraspanin and allows quantative biochemical analyses of the protein complexes by Western blotting and immuno-isolation strategies. Co-culture experiments of dendritic cells with T cells engaging into cognate contacts are used routinely in the lab to measure exosomal transfer. Relative enrichment of the different mGFP tagged tetraspanins which are recruited onto the T cells will be a measure for selective incorporation of the different molecules in dendritic cell exosomes. These analyses will be performed in live cell fluorescence microscopy as well as by FACS analysis.
Molecular cloning, cell culturing, retroviral transduction, biochemical analyses (Western blotting, immuno-precipitation, sucrose-density centrifugation), (live cell) fluorescence microscopy, FACS.
6 or 9 months
R. Wubbolts, R.W.Wubbolts@uu.nl, 030-253 4308
M. Wauben, M.H.M.Wauben@uu.nl, 030-253 5451
Théry C, Ostrowski M, Segura E., (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 9:581
Hemler ME.( 2005) Tetraspanin functions and associated microdomains. Nat Rev Mol Cell Biol. 6:801