Final Report Summary - NANO-ISLANDS (NANOSCALE ANALYSIS OF PROTEIN ISLANDS ON LYMPHOCYTES)
The main objective of our nano-island program was to correct the assumption that most receptors on the cell surface are randomly distributed molecules existing in splendid isolation from each other. We started this project with the finding that IgM and IgD class antigen receptors on mature B lymphocyte are clustered in different class-specific protein islands. A nano-clustering of receptors has recently been described by several other groups but the prevailing idea is that each receptor is clustered on its own. We think that this isolated clustering is not correct but that an array of proteins and lipids come together at nanometer distances to form functional units for which we use the term, nano-islands. Using DNA-coupled chlorotoxin B together with the Fab-based proximity ligation assay (Fab-PLA) we showed that the IgD-BCR resides inside a raft-type lipid compartment whereas the IgM-BCR is associated with these lipids only after B cell activation. Interestingly, many previously described raft-associated proteins such as the co-receptors CD19, CD40, tetraspan proteins including CD20 and chemokine receptors are closely associated with the IgD-BCR whereas the IgM-BCR is associated with the nonraft-associated receptor-phosphatase CD45. We also showed that, upon B cell activation, different nano-islands come together to exchange their protein and lipid content. In this way the IgM-BCR gains access to the CD19/CD20 pro-survival signaling module whereas CD45 moves closer to the IgD-BCR. Such nanoscale re-organization schemes are completely novel and we were among the first groups to describe this. Furthermore, we showed that the nano-island organization of the B lymphocyte membrane is functionally relevant. For example, we found that the chemokine receptor CXCR4 is not only co-clustered together with the IgD-BCR inside the IgD-class nano-island but also functionally dependent on this antigen receptor.
A major question is how nano-islands are generated and maintained on the dynamic plasma membrane. Our finding that caveolin and CD20 are important for the stability of the class-specific nano-island provided a first answer to these questions. Furthermore, we showed that a B cell-specific disturbance of the nano-island organization results in a deregulation of the immune system and autoimmunity. Concerning the generation of nano-islands, we still are working on the nano-island sorting code. Helpful for this would be an efficient proximity proteomics analysis and a catalogue of the composition of all nano-islands on the B lymphocyte membrane, one of the goals that we could not achieve during the ERC funding period. However, we are currently collaborating with a membrane proteomics group and hope to reach this goal in the future. An important game changer for our work is the CRISPR/Cas9 technology that allows us to rapidly delete genes of a human B cell line. We have thus generated many mutant B cell lines and in combination with the Fab-PLA technology this allows us to identify components that are critical for the nano-island organization and stability. Concerning the final goal of our ERC project we studied the alteration of the nano-island organization on several human B cell tumors. We found that B cell chronic lymphocytic leukemia (B-CLL) is associated with an altered nano-island composition and organization resembling more that of activated than of resting B lymphocytes. Furthermore, we identified a novel pro-survival module inside a nano-island that is required for the competitive fitness of a human Burkitt lymphoma cell line. We think that, in the long run, studies of the nano-islands organization on normal or diseased human cells will have a major impact on medical diagnostics and practice.
A major question is how nano-islands are generated and maintained on the dynamic plasma membrane. Our finding that caveolin and CD20 are important for the stability of the class-specific nano-island provided a first answer to these questions. Furthermore, we showed that a B cell-specific disturbance of the nano-island organization results in a deregulation of the immune system and autoimmunity. Concerning the generation of nano-islands, we still are working on the nano-island sorting code. Helpful for this would be an efficient proximity proteomics analysis and a catalogue of the composition of all nano-islands on the B lymphocyte membrane, one of the goals that we could not achieve during the ERC funding period. However, we are currently collaborating with a membrane proteomics group and hope to reach this goal in the future. An important game changer for our work is the CRISPR/Cas9 technology that allows us to rapidly delete genes of a human B cell line. We have thus generated many mutant B cell lines and in combination with the Fab-PLA technology this allows us to identify components that are critical for the nano-island organization and stability. Concerning the final goal of our ERC project we studied the alteration of the nano-island organization on several human B cell tumors. We found that B cell chronic lymphocytic leukemia (B-CLL) is associated with an altered nano-island composition and organization resembling more that of activated than of resting B lymphocytes. Furthermore, we identified a novel pro-survival module inside a nano-island that is required for the competitive fitness of a human Burkitt lymphoma cell line. We think that, in the long run, studies of the nano-islands organization on normal or diseased human cells will have a major impact on medical diagnostics and practice.