Cluster of Excellence –
University of Freiburg

Molecular Immunology

Prof. Dr. Michael Reth

The exact organization of proteins in the membrane of living cells is still poorly understood, but a better knowledge of this topic is of great importance for biological research and medical applications. We have recently developed a quantitative bifluorescent complementation assay (BiFC) and showed with this method that the B cell antigen receptor (BCR) forms auto-inhibitory oligomers on the surface of resting B cells. This discovery lead us to develop the dissociation activation model (DAM) whereby the dissociation and reorganization of BCR oligomers are proposed to be key events during B cell activation. To obtain direct evidence for the proposed BCR dissociation process, we have improved the in situ proximity ligation assay (PLA). In situ PLA detects the close proximity of two target proteins by amplifying a proximity signal using oligo-coupled secondary (2-PLA) or primary (1-PLA) antibodies. The detection limit of the 2-PLA and 1-PLA is 80 and 40 nm, respectively. By conjugating oligos directly to Fab fragments (Fab-PLA), we improved the detection limit of PLA down to 10-20 nm and could directly monitor the dissociation of BCR oligomers on the surface of both murine and human naïve B cells. Currently, we are combining Fab-PLA studies with state-of-art super-resolution microscopy techniques, to investigate the structure, organization and dynamics of the BCR and its interaction with co-receptors. In contrast to the Singer–Nicolson fluid mosaic model, our studies suggest that many membrane proteins are not freely diffusing monomers but rather multicomponent protein complexes pre-organized in nano-size protein islands.