Group Leader

Prof. Dr.  Michael Reth
Prof. Dr. Michael Reth
Adjunct Scientific Member of the MPG (MPI-IE/Uni)
Phone:+49 761 203-2718

Institute of Biology III (Molecular Immunology), University of Freiburg

Michael Reth @ BIOSS Freiburg

Miriam Vitt
Assistant to Michael Reth
Phone:+49 761 203-2868

Laboratory Michael Reth

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Nano-scale organization of the BCR on resting and activated B cells

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.

Upper panel: The schematic drawing shows how the oligomerization of BCR can be monitored by bifluorescent complementation (BiFC) between Igα subunits fused with half domains of fluorescent proteins. Lower panel: BiFC signals are detected on the surface of B cells expressing BCRs containing the tagged Igα subunits by confocal microscopy. Zoom Image

Upper panel: The schematic drawing shows how the oligomerization of BCR can be monitored by bifluorescent complementation (BiFC) between Igα subunits fused with half domains of fluorescent proteins. Lower panel: BiFC signals are detected on the surface of B cells expressing BCRs containing the tagged Igα subunits by confocal microscopy.

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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.

 
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