Prof. Dr. Robert Tampé
Goethe University, Institute of Biochemistry, Cellular Biochemistry
1) Cellular logistics: In-situ photo-confinement of membrane receptors
Cell-cell communication relies on the dynamic assembly of receptor-ligand complexes at the plasma membrane. Receptor clustering is a key process in signal transduction and pleiotropic downstream cell responses. Heterotrimeric G protein-coupled receptors (GPCRs) are members of a large family of membrane proteins that mediate a myriad of cellular processes. The physiological relevance of GPCR clustering or confinement is, however, poorly understood. In this proposal, we aim to control the membrane organization of receptors and to reveal how the location, lateral diffusion and density of receptor confinement modulates early downstream signaling events and the final physiological outcome. By an integrative approach using photo-activatable cell matrices, ultra-small lock-and-key elements, and G-protein sensors, we will study receptor clustering in-situ on living-cells. We aim to decode the mechanism underlying the cellular logistics of GPCR activation by confinement, downstream signaling, and receptor-mediated mechanotransduction.
1) Klein et al. (2018) Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies. Chem Science, 9, 7835. doi:10.1039/c8sc02910e
2) Gatterdam K, Joest EF, Dietz MS, Heilemann M, Tampé R (2018) Super-chelators for advanced protein labeling in living cells. Angew Chem Int Ed 57, 5620-5. doi:10.1002/anie.201800827
3) Guesdon et al. (2016) EB1 interacts with outwardly curved and straight regions of the microtubule lattice. Nature Cell Biology 8, 1102-8. doi:10.1038/ncb3412
4) Kollmannsperger A, Sharei A, Raulf A, Heilemann M, Langer R, Jensen KF, Wieneke R, Tampé R (2016) Live-cell protein labelling with nanometre precision by cell squeezing.
Nature Communications 7, 10372. doi:10.1038/ncomms10372
2) Nanocluster assembly of MHC I peptide-loading complexes in dendritic cells
Dendritic cells (DCs) translate local innate responses into long-lasting adaptive immunity by priming antigen-specific T cells. Accordingly, there is an ample interest in exploiting DCs for therapeutic purposes, e.g. in personalized immunotherapies. Despite recent advances in elucidating molecular pathways of antigen processing, their exact spatial organization in DCs is largely unknown. In this project we aim to elucidate the subcellular distribution of the TAP-dependent antigen processing machinery in human monocyte-derived DCs by next-generation protein-protein interaction anaylsis, CRISPR/Cas9 gene editing in primary human cells, super-resolution microscopy and electron tomography. We plan to uncover the subcellular organization of antigen processing machineries, which is basically unknown. Independent of the DC activation status, peptide-loading complexes (PLCs) are organized in nanoclusters comprised of several PLCs. On the nanometer scale, PLCs accumulate along a tubular ER network that is highly compressed in the tips of the dendrites. Our data indicate subcellular modifications of DCs that are associated with their T-cell stimulatory capacity, paving the way towards more effective DC-based therapies.
1) Döring et al. (2019) Modulation of TAP-dependent antigen compartmentalization during human monocyte-to-DC differentiation. Blood Adv 3, 839-50. (*corr. author) doi:10.1182/bloodadvances.2018027268
2) Thomas C, Tampé R (2019) MHC I chaperone complexes shaping immunity.
Curr Opin Immunol 58, 9-15. doi:10.1016/j.coi.2019.01.001
3) Blees et al. (2017) Structure of the human MHC-I peptide-loading complex. Nature 551, 525-8.
4) Thomas C, Tampé R (2017) Structure of the TAPBPR-MHC I complex defines the mechanism of peptide loading and editing. Science 358, 1060-4. doi:10.1126/science.aao6001
5) Fischbach et al. (2015) Ultra-sensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets. Nat Commun 6, 6199. doi:10.1038/ncomms7199