Regulation of B Cell Function by Membrane-Cytoskeletal Remodeling

通过膜细胞骨架重塑调节 B 细胞功能

• 批准号: 8085871
• 负责人: Neetu Gupta
• 金额: $ 34.62万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-25 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8085871
• 关键词: Actins; Affect; Affinity Chromatography; Antibodies; Antigen Presentation; Antigens; Architecture; Aspartate; Autoimmune Diseases; B-Cell Activation; B-Cell Development; B-Lymphocytes; Bacteria; Binding; Binding Proteins; Binding Sites; Cell membrane; Cell physiology; Cells; Comprehension; Cytoskeleton; Dendritic Cells; Disease; Event; Exhibits; Family; Hematopoietic; Homeostasis; Image; Immune; Immune response; Immunity; Infection; Isotopes; Lead; Learning; Ligation; Link; Location; Lymphoid Tissue; Mediating; Membrane; Memory; Microscopy; Modification; Molecular; Molecular Conformation; Mus; Mutate; Mutation; Nature; Output; Phosphorylation; Phosphorylation Site; Protein Dephosphorylation; Proteins; Reaction; Receptors, Antigen, B-Cell; Regulation; Relative (related person); Reporting; Research; Rheumatoid Arthritis; Role; Signal Pathway; Signal Transduction; Skeleton; Spatial Distribution; Stimulus; Stromal Cell-Derived Factor 1; Structure; Structure-Activity Relationship; Surface; Systemic Lupus Erythematosus; T-Lymphocyte; Testing; Therapeutic; Threonine; Time; Tyrosine; Vaccination; Virus; base; cell behavior; cell motility; chemokine; crosslink; design; early childhood; ezrin; flexibility; human disease; in vivo; insight; member; migration; moesin; mutant; novel; pathogen; public health relevance; radixin protein; response; spatiotemporal

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：B细胞通过在质膜上组装信号体来响应环境抗原以进行信号转导。它们在整个体内迁移以进行监测，并与T细胞和淋巴组织中的树突状细胞相互作用，以进行抗原表现。所有这些事件都与B细胞膜的动态形态和组成重组有关。膜重塑需要从潜在的皮质细胞骨架中耦合膜，但对其对B细胞功能的调节和影响很少了解。我们先前已经报道说，Ezrin是Ezrin-radixin-Moesin（ERM）家族的成员，调节膜 - 骨架骨骼解偶联，以响应B细胞抗原受体（BCR）刺激。 Ezrin通过在其肌动蛋白结合位点的临界苏氨酸残基（T567）处进行去磷酸化来实现这一目标，从而导致折叠结构，而折叠结构无能力进行膜 - 环骨架交联。我们观察到用趋化因子SDF-11处理B细胞后，T567的去磷酸化相似。 T567的磷酸化突变是天冬氨酸，使Ezrin保持开放构象，导致抑制BCR依赖性膜动力学和SDF-111依赖性B细胞迁移。有条件的Ezrin缺乏小鼠的B细胞也表现出严重受损的迁移。因此，ezrin的缺失以及ezrin结构的操纵都会影响B细胞的迁移，这表明完整的Ezrin结构对于B细胞迁移是必不可少的。有趣的是，BCR而不是SDF-11刺激会诱导Ezrin对独特的酪氨酸残基的稳健磷酸化，这表明Ezrin参与BCR连接下游的信号转导。我们假设Ezrin通过经过多种和差异的磷酸化和去磷酸化反应来响应抗原和趋化因子来策划B细胞的激活和迁移。这些修饰可能定义了Ezrin根据刺激的性质提供束缚或信号功能的能力。我们建议使用两种情况测试Ezrin在调节B细胞功能中的作用，首先，B细胞异位表达Ezrin的磷酸化位点突变体，其次是B细胞源自Ezrin条件缺失的小鼠。以下特定目标旨在检验我们的假设。 （1）测试Ezrin磷酸化在B细胞激活和迁移中的作用，（2），以阐明Ezrin定位的时空动力学和B细胞激活和迁移过程中的相互作用的时空动力学，以及（3）以研究B细胞激活和VIVO中EZRIN在B细胞激活和迁移中的功能。我们提出的对Ezrin缺陷小鼠的B细胞和Ezrin的磷酸化位点突变体的研究将提供有关Ezrin和B细胞行为之间结构功能关系的见解。由于在所有造血细胞中均表达Ezrin，因此从我们的研究中学到的经验教训可以应用于对感染和免疫期间其他免疫细胞动力学的更广泛理解。最终，我们的研究将促进对信号通路和细胞结构扰动产生的免疫疾病的更好理解。公共卫生相关性：B细胞通过分泌清除感染的特定抗体来应对细菌和病毒等环境病原体。这为在儿童早期疫苗接种期间产生的记忆提供了基础。故障B细胞与自身免疫性疾病（如全身性红斑狼疮和类风湿关节炎）有关。为了有效发挥作用，B细胞需要在淋巴组织中迁移以进行监测，并且在结合致病蛋白后，需要重新组织其表面成分以进行激活。我们假设Ezrin是一种蛋白质，可帮助B细胞实现其功能。 Ezrin具有将细胞膜连接到细胞骨骼的能力，并可能调节B细胞的分子重组和迁移。我们建议通过在B细胞中突变或在B细胞中删除EZRIN在B细胞功能中的作用并测试对B细胞激活和迁移的影响。我们还建议鉴定Ezrin结合的蛋白质及其在激活过程中其细胞位置。从了解埃兹林在B细胞功能中的作用中学到的经验教训可以应用于B细胞依赖性人类疾病的治疗设计。