Regulation of B Cell Function by Membrane-Cytoskeletal Remodeling

描述（由申请方提供）：B细胞通过在质膜上组装信号体进行信号转导来响应环境抗原。它们在全身迁移以进行监视，并与淋巴组织内的T细胞和树突状细胞相互作用以进行抗原呈递。所有这些事件都与B细胞膜的动态形态和组成重组有关。膜重塑需要膜从底层皮质细胞骨架解偶联，但其对B细胞功能的调节和影响知之甚少。我们以前曾报道，ezrin，ezrin-radixin-moesin（ERM）家族的成员调节细胞骨架解偶联反应B细胞抗原受体（BCR）刺激。Ezrin通过在其肌动蛋白结合位点的关键苏氨酸残基（T567）处进行脱磷酸化来实现这一点，这导致不能进行膜-细胞骨架交联的折叠结构。在用趋化因子SDF-11处理B细胞后，我们观察到T567的类似去磷酸化。将T567磷酸化突变为天冬氨酸，使ezrin保持开放构象，导致抑制BCR依赖性膜动力学和SDF-11依赖性B细胞迁移。来自条件性ezrin缺陷小鼠的B细胞也表现出严重受损的迁移。因此，ezrin的缺乏以及ezrin结构的操纵都会影响B细胞迁移，这表明完整的ezrin结构对于B细胞迁移是必需的。有趣的是，BCR而不是SDF-11刺激诱导ezrin在独特的酪氨酸残基上的强烈磷酸化，表明ezrin参与BCR连接下游的信号转导。我们假设ezrin通过对抗原和趋化因子的反应进行多重和差异的磷酸化和去磷酸化反应来协调B细胞的活化和迁移。这些修饰可能定义了埃兹蛋白根据刺激的性质提供束缚或信号功能的能力。我们建议使用两种方案来测试ezrin在调节B细胞功能中的作用，第一种方案是B细胞异位表达ezrin的磷酸化位点突变体，第二种方案是B细胞来源于ezrin有条件缺失的小鼠。以下具体目标旨在检验我们的假设。具体目的是（1）测试ezrin磷酸化在B细胞活化和迁移中的作用，（2）阐明ezrin在B细胞活化和迁移过程中的定位和相互作用的时空动态，以及（3）研究ezrin在体内B细胞活化和迁移中的功能。我们提出的研究与B细胞从ezrin缺陷小鼠和磷酸化位点突变体ezrin将提供深入了解ezrin和B细胞行为之间的结构-功能关系。由于埃兹蛋白在所有造血细胞中表达，从我们的研究中吸取的教训可以应用于更广泛地了解感染和免疫过程中其他免疫细胞的动态。最终，我们的研究将有助于更好地理解由信号通路和细胞结构扰动引起的免疫疾病。公共卫生相关性：B细胞通过分泌清除感染的特异性抗体对环境病原体如细菌和病毒作出反应。这为儿童早期接种疫苗期间产生的记忆提供了基础。功能障碍的B细胞与系统性红斑狼疮和类风湿性关节炎等自身免疫性疾病有关。为了有效地发挥功能，B细胞需要在淋巴组织中迁移以进行监视，并且在结合致病蛋白时，它们需要重组其表面组分以进行活化。我们假设埃兹蛋白是一种帮助B细胞完成其功能的蛋白质。Ezrin具有将细胞膜连接到细胞骨架的能力，并且可以调节B细胞的分子重组和迁移。我们建议通过在B细胞中突变或删除埃兹蛋白并测试其对B细胞活化和迁移的影响来测试埃兹蛋白在B细胞功能中的作用。我们还建议确定蛋白质的ezrin结合，其细胞位置在激活过程中。从理解埃兹蛋白在B细胞功能中的作用中获得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。

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.