The regulation of signaling and cytoskeletal rearrangements in B-lymphocytes

B 淋巴细胞信号传导和细胞骨架重排的调节

• 批准号: RGPIN-2019-04911
• 负责人: Matsuuchi, Linda
• 金额: $ 2.33万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737947
• 关键词: regulation; signaling; cytoskeletal; rearrangements; lymphocytes

Interactions among signaling pathways that link antigen receptors to remodeling of the actin cytoskeleton are not completely understood. The initiation of signaling involves receptors that nucleate the formation of "signalosomes" that contain adaptor proteins, kinases, phosphatases, and their targets. Previous work described and analyzed the role of the B-cell antigen receptor (BCR) as the key signaling receptor that regulates B-lymphocyte (B cell) activation, development, and antigen gathering, responses requiring actin rearrangements. We discovered that the gap junction protein connexin43 (Cx43) that forms channels between cells and the environment, also has key non-channel functions, due to its unique carboxyl-tail (CT). These roles include regulating the actin cytoskeleton, through mechanisms that are unclear. Our goal is to understand how receptor signaling is coupled to scaffolding/adaptor proteins that regulate actin rearrangements, using B cells as a model. We will test the hypothesis that BCR signaling is influenced by Cx43 expression and that they both work collaboratively with interacting proteins to regulate cytoskeletal responses. Aim 1. To determine how Cx43 effects BCR signaling. Aim 2. To determine how the BCR and Cx43 are organized at the plasma membrane. Aim 3. To determine the effects of the cytoplasmic tail (CT) of Cx43 on rearrangements of the cytoskeleton in B cells. We propose that Cx43 acts as a scaffolding or adaptor protein to regulate either receptor organization in the membrane, or in the process of successive breakdown and rearrangement of actin to promote changes in the cytoskeleton. We have used a genetic mutational approach to identify important amino acids in the Cx43 CT and made a series of deletions and point mutations, many normally phosphorylated.  We have defined the region of the Cx43 CT between amino acids 246 to 307 that is necessary for BCR-mediated cell spreading in our model, as well as identified critical tyrosines and serines in the CT that when mutated inhibit cytoskeletal rearrangements. We will focus on a subset of the mutants. We predict that these targeted residues in the CT are sites of critical protein interactions that are nucleated by Cx43 and link receptors to the machinery that controls cytoskeletal architecture. Since cytoskeletal rearrangements are common to all cell types, we propose that we will gain an overall understanding of the complex interplay between similar families of proteins and the actin cytoskeletal network. In addition to B cells, Cx43 has been shown to be important for neuronal cell migration, cardiac and artery development, and invasiveness of cancer cells during tumor progression. The common features of Cx43's influence in different cell and organ systems suggests that it is involved in fundamental mechanisms that underlie cytoskeletal functions, and our studies will reveal interactions that are important for these basic effects.

将抗原受体与肌动蛋白细胞骨架重塑联系起来的信号通路之间的相互作用尚不完全清楚。信号传导的启动涉及受体，这些受体使“信号小体”的形成成核，其中包含衔接蛋白、激酶、磷酸酶及其靶标。先前的工作描述并分析了 B 细胞抗原受体 (BCR) 作为调节 B 淋巴细胞 (B 细胞) 激活、发育和抗原收集以及需要肌动蛋白重排的反应的关键信号传导受体的作用。我们发现，间隙连接蛋白 connexin43 (Cx43) 在细胞和环境之间形成通道，由于其独特的羧基尾 (CT)，也具有关键的非通道功能。这些作用包括通过尚不清楚的机制调节肌动蛋白细胞骨架。我们的目标是以 B 细胞为模型，了解受体信号传导如何与调节肌动蛋白重排的支架/适配器蛋白偶联。我们将测试这样的假设：BCR 信号传导受 Cx43 表达的影响，并且它们都与相互作用的蛋白质协同作用来调节细胞骨架反应。目标 1. 确定 Cx43 如何影响 BCR 信号传导。 目标 2. 确定 BCR 和 Cx43 在质膜上的组织方式。 目标 3. 确定 Cx43 的细胞质尾 (CT) 对 B 细胞中细胞骨架重排的影响。 我们认为 Cx43 作为支架或衔接蛋白来调节膜中的受体组织，或者在肌动蛋白的连续分解和重排过程中调节以促进细胞骨架的变化。我们使用基因突变方法来鉴定 Cx43 CT 中的重要氨基酸，并进行了一系列缺失和点突变，其中许多通常是磷酸化的。  我们已经在我们的模型中定义了 Cx43 CT 氨基酸 246 至 307 之间的区域，这是 BCR 介导的细胞扩散所必需的，并确定了 CT 中的关键酪氨酸和丝氨酸，当突变时，它们会抑制细胞骨架重排。我们将重点关注突变体的一个子集。我们预测 CT 中的这些目标残基是由 Cx43 成核的关键蛋白质相互作用位点，并将受体与控制细胞骨架结构的机制连接起来。由于细胞骨架重排对于所有细胞类型都是常见的，因此我们建议我们将对相似蛋白质家族和肌动蛋白细胞骨架网络之间复杂的相互作用有一个全面的了解。除了 B 细胞外，Cx43 已被证明对神经元细胞迁移、心脏和动脉发育以及肿瘤进展过程中癌细胞的侵袭性也很重要。 Cx43 在不同细胞和器官系统中影响的共同特征表明，它参与了细胞骨架功能的基本机制，我们的研究将揭示对这些基本效应很重要的相互作用。