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Biochemistry and Cell Biology

Tuesday 5 May 2009

Prof.dr W. Stoorvogel and dr R. Wubbolts

Project
Project
Dendritic cells (DCs) are antigen presenting cells that are most potent in generating primary immune responses. MHC II molecules are key components in the initiation of these cellular immunological responses. DC populations in peripheral tissues are efficient in sampling the environment by a highly efficient endocytic system. These cells retain MHC II molecules actively in their endosomal system. When DCs are stimulated by pathogen derivatives (thereby activating patterning receptors, i.e. TLRs) this retention is lifted and MHC II molecules are shuttled to the cell surface. We are interested in the molecular mechanisms that drive these developmental changes in DCs. A major event in this process is the conjugation of ubiquitin molecules to the cytoplasmic tail of MHC II. This sorting motif drives sorting of MHCII to lysosomes. Ubiquitination of MHC II in activated DC is blocked thereby retaining the molecules on the plasma membrane. Key questions in our research are focused on the timing and regulation of these ubiquitin modifications in immune modulatory conditions.

Project 1: Determine timing and localization of ubiquitin modification after generic DC stimulation protocols with cytokines and immunomodulatory factors (LPS, IL-10, dexamethason) and by subcellular restricted simulation provided by phagocytosis of functionalized beads.

Project 2: Study the role of the ubiquitin ligase MARCH-1 and the effect of another motif in the MHC II cytoplasmic tail (di-leucine) on MHC II sorting when DCs mature. GFP tagging of the ligase and molecular modification of motifs in the MHC II tails allow these analyses.

Techniques
Fluorescence imaging of live cells, molecular cloning, siRNA transfection, retroviral transduction, stable cell line generation, biochemical analyses, phagosome/organelle isolation, antigen presentation assays, FACS

Duration
6 or 9 months

Contact
Richard Wubbolts, R.W.Wubbolts@uu.nl, 030-253 4308
Toine ten Broeke, W.Stoorvogel@uu.nl, 030-253 2454

References
- van Niel G, Wubbolts R, Stoorvogel W. (2008) Endosomal sorting of MHC class II determines antigen presentation by dendritic cells. Curr Opin Cell Biol. 20, 437
- van Niel G, Wubbolts R, Ten Broeke T, Buschow SI, Ossendorp FA, Melief CJ, Raposo G, van Balkom BW, Stoorvogel W. (2006) Dendritic cells regulate exposure of MHC class II at their plasma membrane by oligoubiquitination. Immunity. 25, 885
- ten Broeke T, de Graaff A, van 't Veld EM, Wauben MHM, Stoorvogel W and Wubbolts R (2009) Trafficking of MHC Class II in Dendritic Cells is Dependent on but Not Regulated by Degradation of Its Associated Invariant Chain Traffic 1, 324


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