Regulation of B Cell Function by Membrane-Cytoskeletal Remodeling
Principal Investigator: Neetu Gupta
Abstract: B cells respond to environmental antigens by assembling signalosomes at the plasma membrane for signal transduction. They migrate throughout the body for surveillance, and interact with T cells and dendritic cells within lymphoid tissue for antigen presentation. All these events are associated with dynamic morphological and compositional reorganization of the B cell membrane. Membrane remodeling requires membrane uncoupling from the underlying cortical cytoskeleton but its regulation and impact on B cell function is poorly understood. We have previously reported that ezrin, a member of the ezrin-radixin-moesin (ERM) family regulates membrane-cytoskeletal uncoupling in response to B cell antigen receptor (BCR) stimulation. Ezrin accomplishes this by undergoing dephosphorylation at a critical threonine residue (T567) in its actin-binding site which results in a folded structure that is incapable of membrane-cytoskeletal crosslinking. We observed a similar dephosphorylation of T567 upon treatment of B cells with the chemokine SDF-11. A phosphomimetic mutation of T567 to aspartate that holds ezrin in an open conformation, results in inhibition of BCR-dependent membrane dynamics and SDF-11-dependent B cell migration. B cells from conditional ezrin-deficient mice also exhibit severely impaired migration. Thus, both the absence of ezrin as well as manipulation of ezrin structure affects B cell migration, suggesting that an intact ezrin structure is necessary for B cell migration. Interestingly, BCR, but not SDF-11 stimulation induces robust phosphorylation of ezrin on unique tyrosine residues suggesting that ezrin participates in signal transduction downstream of BCR ligation. We hypothesize that ezrin orchestrates B cell activation and migration by undergoing multiple and differential phosphorylation and dephosphorylation reactions in response to antigen and chemokines. These modifications likely define ezrin's ability to provide a tethering or signaling function depending on the nature of the stimulus. We propose to test the role of ezrin in regulating B cell function using two scenarios, first in which B cells ectopically express phosphorylation site mutants of ezrin, and second in which B cells are derived from mice with conditional deletion of ezrin. The following specific aims are designed to test our hypotheses. The specific aims are (1) to test the role of ezrin phosphorylation in B cell activation and migration, (2) to elucidate the spatiotemporal dynamics of ezrin's localization and interactions during B cell activation and migration, and (3) to investigate the function of ezrin in B cell activation and migration in vivo. Our proposed studies with B cells from ezrin-deficient mice and phosphorylation site mutants of ezrin will provide insights into structure-function relationships between ezrin and B cell behavior. Since ezrin is expressed in all hematopoietic cells, lessons learnt from our studies could be applied towards a broader understanding of the dynamics of other immune cells during infection and immunity. Ultimately, our research will facilitate better comprehension of immunological disorders that result from perturbation of signaling pathways and cellular architecture. Public Health Relevance: B cells respond to environmental pathogens such as bacteria and viruses by secreting specific antibodies that clear infections. This provides the basis for memory that is generated during early childhood vaccinations. Malfunctioning B cells are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. To function effectively, B cells need to migrate in the lymphoid tissues for surveillance, and upon binding pathogenic proteins they need to reorganize their surface components for activation. We hypothesize that ezrin is a protein that helps B cells in accomplishing their function. Ezrin has the ability to link the cell membrane to the cellular skeleton, and may regulate the molecular reorganization and migration of B cells. We propose to test the role of ezrin in B cell function by mutating it or deleting it in B cells and testing the impact on B cell activation and migration. We also propose to identify the proteins that ezrin binds to, and its cellular location during activation. Lessons learnt from understanding the role of ezrin in B cell function can be applied towards therapeutic design for B cell-dependent human diseases.
Funding Period: 2009-07-25 - 2014-06-30
more information: NIH RePORT