Mechanisms of Node of Ranvier Assembly

Ranvier组装节点的机制

• 批准号: 10212457
• 负责人: JAMES SALZER
• 金额: $ 46.98万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10212457
• 关键词: ANK3 gene; Actins; Action Potentials; Agreement; Axon; Binding; Biology; Caliber; Cell Adhesion Molecules; Clathrin; Coculture Techniques; Complex; Cytoskeleton; Diffuse; Disease; Dynamin I; Endocytosis; Engineering; Extranodal; Fiber; Future; Generations; Hippocampus (Brain); In Vitro; Knock-out; Lead; Length; Macromolecular Complexes; Maintenance; Mediating; Modeling; Morphology; Mus; Myosin Heavy Chains; Myosin Light Chains; Myosin Regulatory Light Chains; Myosin Type II; Natural regeneration; Nervous System Physiology; Neuraxis; Neuroglia; Neurons; Nodal; Pathogenesis; Peripheral; Potassium; Proteins; Proteome; Ranvier' s Nodes; Recovery of Function; Reporting; Research; Role; Signal Transduction; Site; Sodium; Surface; Testing; Transgenic Mice; base; betaIV spectrin; conditional knockout; in vivo; insight; knock-down; loss of function; mutant; myelination; nervous system disorder; nodal protein; non-muscle myosin; novel; novel therapeutic intervention; recruit; repaired; scaffold; therapy development; voltage

The axon initial segment (AIS) and nodes of Ranvier are sites of action potential generation and regeneration, respectively and are thus critical for the proper function of the nervous system. Their key roles in electrogenesis results from the striking enrichment of a macromolecular complex of voltage-gated sodium (NaV) and potassium (KCNQ) channels, cell adhesion molecules (NF186, NrCAM), and a cytoskeletal scaffold of ankyrin G (AnkG) and beta-IV (βIV) spectrin. Prior to myelination, nodal components are diffusely distributed along axons consistent with their continuous conduction of action potentials. With myelination, the axon reorganizes into discrete domains, culminating in node assembly, thus enabling saltatory conduction. A key question is what drives this reorganization? We have found two complementary mechanisms broadly contribute: i) recruitment signals that target and stabilize this complex at nodes and ii) active clearance that removes nodal proteins from everywhere else along the axon. Here, we examine the contribution of both mechanisms to node formation. Recruitment signals at PNS and CNS nodes culminate in assembly of an AnkG/βIV spectrin cytoskeleton scaffold to which all other nodal components bind. This scaffold is further tethered to and likely stabilized by regularly spaced, sub-membranous actin rings at both the AIS and nodes. We recently reported actin rings at the AIS and nodes are specifically modified by contractile myosin II. In particular, phosphorylated myosin light chain (pMLC) - the regulatory subunit that activates the contractile function of myosin II - is enriched at and an early marker of the AIS and nodes. Strategies that increase or decrease pMLC levels/myosin II activity, drive AIS assembly and disassembly, respectively. These results implicate contractile NMII as a novel regulator of the AIS and suggest a conserved role at nodes, a notion strongly supported by MLC knockdown studies. We have also found that just prior to myelination, glial cells drive clearance of nodal components from the internode by clathrin-mediated endocytosis (CME). Our results suggest cleared proteins are not linked to the cytoskeleton and can therefore be clustered for endocytosis. In agreement expression of a mutant NF186 construct engineered to bind to the internodal cytoskeleton, is not endocytosed but rather is persistently expressed along the axon. Strikingly, this construct delays myelination. This latter finding suggests clearance has dual roles in sculpting the node and preparing the axon for myelination thereby coordinating assembly of the node of Ranvier with myelination of axons. Here, we test key aspects of this model, including the role of MLC/myosin II in node assembly/stability and actin ring integrity by knockdown and knockout strategies. We will also examine the mechanisms and consequences of this glia-driven clearance of axonal proteins, including modeling defective CME in mice to broadly perturb the axon surface proteome and assess its effects on myelination. These studies will provide important new insights into axo-glial interactions that regulate node formation and myelination and, potentially, into pathogenetic mechanisms that contribute to disorders of myelinated fibers.

轴突初始段（AIS）和朗飞结是动作电位产生和再生的部位， 分别，因此对于神经系统的正常功能至关重要。它们在电发生中的关键作用 电压门控钠 (NaV) 和钾的大分子复合物显着富集的结果 (KCNQ) 通道、细胞粘附分子 (NF186、NrCAM) 和锚蛋白 G 的细胞骨架支架 (AnkG) 和 β-IV (βIV) 血影蛋白。在髓鞘形成之前，节点成分沿轴突广泛分布 与他们的动作电位的连续传导一致。随着髓鞘形成，轴突重组为 离散域，最终形成节点组装，从而实现跳跃传导。一个关键问题是什么 推动这次重组？我们发现两种互补机制具有广泛的贡献：i) 招募 靶向并稳定节点处该复合物的信号，以及 ii) 主动清除，从节点中去除节点蛋白 沿着轴突的其他地方。在这里，我们研究了两种机制对节点形成的贡献。 PNS 和 CNS 节点的募集信号最终形成 AnkG/βIV 血影蛋白细胞骨架支架 所有其他节点组件都与其绑定。该支架进一步固定并可能通过定期固定 AIS 和节点处都有间隔的膜下肌动蛋白环。我们最近报道了 AIS 的肌动蛋白环和 节点由收缩性肌球蛋白 II 专门修饰。特别是磷酸化肌球蛋白轻链 (pMLC) - 激活肌球蛋白 II 收缩功能的调节亚基 - 在以下位置富集并是其早期标志物 AIS 和节点。增加或减少 pMLC 水平/肌球蛋白 II 活性、驱动 AIS 组装和 分别进行拆卸。这些结果表明收缩性 NMII 是 AIS 的一种新型调节器，并表明 MLC 敲除研究强烈支持这一概念。我们还发现 在髓鞘形成之前，神经胶质细胞通过网格蛋白介导从节间清除节点成分 内吞作用（CME）。我们的结果表明，清除的蛋白质与细胞骨架无关，因此可以 聚集进行内吞作用。突变体 NF186 构建体的一致表达被设计为结合 节间细胞骨架不被内吞，而是沿着轴突持续表达。引人注目的是，这 构建延迟髓鞘形成。后一个发现表明间隙在雕刻节点和 准备轴突的髓鞘形成，从而协调朗飞结的组装与髓鞘形成 轴突。在这里，我们测试了该模型的关键方面，包括 MLC/肌球蛋白 II 在节点组装/稳定性中的作用 通过敲低和敲除策略实现肌动蛋白环的完整性。我们还将研究机制和 这种神经胶质细胞驱动的轴突蛋白清除的后果，包括在小鼠中模拟有缺陷的 CME 广泛干扰轴突表面蛋白质组并评估其对髓鞘形成的影响。这些研究将提供 关于调节节点形成和髓鞘形成的轴-神经胶质相互作用的重要新见解，并且有可能 探究导致有髓纤维疾病的发病机制。