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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=766130
• 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未完全了解。信号传导的起始涉及使“信号体”的形成成核的受体，所述“信号体”含有衔接蛋白、激酶、磷酸酶及其靶点。以前的工作描述和分析了B细胞抗原受体（BCR）作为调节B淋巴细胞（B细胞）活化、发育和抗原聚集的关键信号受体的作用，这些反应需要肌动蛋白重排。我们发现在细胞和环境之间形成通道的差距连接蛋白连接蛋白43（Cx43）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，了解受体信号是如何与调节肌动蛋白重排的支架/衔接蛋白相结合的。我们将测试的假设，即BCR信号的影响Cx43的表达，他们都与相互作用的蛋白质，以调节细胞骨架的反应。目标1。探讨Cx43对BCR信号通路的影响。 目标2.确定BCR和Cx43如何在质膜上组织。 目标3。目的：研究Cx43胞质尾区（CT）对B细胞骨架重排的影响。 我们建议，Cx43作为一个支架或适配器蛋白调节受体组织在膜上，或在连续的分解和重排的肌动蛋白，以促进细胞骨架的变化的过程中。我们已经使用了遗传突变的方法来确定重要的氨基酸在Cx43 CT和一系列的缺失和点突变，许多正常磷酸化。 我们已经确定了Cx43 CT的氨基酸246至307之间的区域，该区域是我们模型中BCR介导的细胞扩散所必需的，并且确定了CT中突变时抑制细胞骨架重排的关键酪氨酸和丝氨酸。我们将集中在变种人的一个子集。我们预测，CT中的这些靶向残基是关键蛋白质相互作用的位点，这些蛋白质相互作用由Cx43成核，并将受体连接到控制细胞骨架结构的机制。由于细胞骨架重排是常见的所有类型的细胞，我们建议，我们将获得一个全面的了解相似的蛋白质家族和肌动蛋白细胞骨架网络之间的复杂的相互作用。除了B细胞之外，Cx43已被证明对于神经元细胞迁移、心脏和动脉发育以及肿瘤进展期间癌细胞的侵袭性是重要的。Cx43在不同细胞和器官系统中的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作用重要的相互作用。