Regulation of B Cell Function by Membrane-Cytoskeletal Remodeling

描述(申请人提供)：B细胞对环境抗原的反应是通过在质膜上组装信号小体进行信号转导。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呈递抗原。所有这些事件都与B细胞膜的动态形态和成分重组有关。膜重塑需要从底层的皮质细胞骨架解偶联，但它的调节和对B细胞功能的影响还知之甚少。我们之前已经报道，Ezrin是Ezrin-Radisin-moesin(ERM)家族的成员，它调节B细胞抗原受体(BCR)刺激下的膜-细胞骨架解偶联。Ezrin通过在其肌动蛋白结合部位的关键苏氨酸残基(T567)上进行去磷酸化来实现这一点，这导致了不能膜-细胞骨架交联的折叠结构。我们观察到在用趋化因子SDF-11处理B细胞时，T567也发生了类似的去磷酸化。T567的天冬氨酸拟磷突变使Ezrin处于开放构象，导致依赖BCR的膜动力学和依赖SDF-11的B细胞迁移受到抑制。条件性Ezrin缺陷小鼠的B细胞也表现出严重的迁移障碍。因此，Ezrin的缺失以及Ezrin结构的调控都会影响B细胞的迁移，这表明完整的Ezrin结构是B细胞迁移所必需的。有趣的是，BCR而不是SDF-11刺激诱导了Ezrin在独特的酪氨酸残基上的强烈磷酸化，这表明Ezrin参与了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经历多次和不同的磷酸化和去磷酸化反应来协调B细胞的激活和迁移。这些修改可能定义了Ezrin提供系绳或信号传递功能的能力，这取决于刺激的性质。我们建议使用两种情况来测试Ezrin在调节B细胞功能中的作用，第一种情况是B细胞异位表达Ezrin的磷酸化位点突变，第二种情况是B细胞来自条件缺失Ezrin的小鼠。以下具体目标旨在检验我们的假设。其具体目的是(1)检测Ezrin磷酸化在B细胞激活和迁移中的作用；(2)阐明Ezrin在B细胞激活和迁移过程中的定位和相互作用的时空动力学；(3)研究Ezrin在体内B细胞激活和迁移中的作用。我们建议对Ezrin缺陷小鼠的B细胞和Ezrin的磷酸化位点突变体进行的研究将为Ezrin和B细胞行为之间的结构-功能关系提供深入的见解。由于Ezrin在所有造血细胞中都有表达，从我们的研究中学到的经验可以用于更广泛地理解其他免疫细胞在感染和免疫过程中的动态。最终，我们的研究将有助于更好地理解由于信号通路和细胞结构的扰动而导致的免疫紊乱。公共卫生相关性：B细胞对细菌和病毒等环境病原体的反应是通过分泌特定的抗体来清除感染。这为儿童早期接种疫苗期间产生的记忆提供了基础。B细胞功能障碍与自身免疫性疾病有关，如系统性红斑狼疮和类风湿性关节炎。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，并且在与致病蛋白结合后，它们需要重组其表面成分以进行激活。我们假设Ezrin是一种帮助B细胞完成其功能的蛋白质。Ezrin具有连接细胞膜和细胞骨架的能力，可能调节B细胞的分子重组和迁移。我们建议通过在B细胞中突变或删除Ezrin来测试它在B细胞功能中的作用，并测试它对B细胞激活和迁移的影响。我们还建议确定Ezrin结合的蛋白质，以及它在激活过程中的细胞位置。从了解Ezrin在B细胞功能中的作用中学到的经验可以应用于B细胞依赖型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.