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.

轴突起始段（AIS）和Ranvier结是动作电位产生和再生的部位， 因此对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 这是由于电压门控钠（NaV）和钾的大分子复合物的显著富集 （KCNQ）通道、细胞粘附分子（NF 186、NrCAM）和锚蛋白G（AnkG）的细胞骨架支架 和β-IV（βIV）血影蛋白。在髓鞘形成之前，结节成分沿着轴突弥散分布 与它们动作电位的连续传导相一致。随着髓鞘的形成，轴突重组成 离散域，最终在节点组装，从而使跳跃式传导。一个关键问题是什么 推动这一重组？我们发现两种互补机制广泛地起作用： i）在节点处靶向并稳定该复合物的信号，和ii）从细胞中去除节点蛋白的主动清除， 沿着轴突的其他地方。在这里，我们研究这两种机制的节点形成的贡献。 PNS和CNS节点的募集信号最终导致AnkG/βIV血影蛋白细胞骨架支架的组装 所有其他节点组件都绑定到它。该支架被进一步拴系到并可能通过定期固定而稳定。 间隔，亚膜肌动蛋白环在AIS和节点。我们最近报道了AIS的肌动蛋白环， 淋巴结被收缩性肌球蛋白II特异性修饰。特别地，磷酸化肌球蛋白轻链（pMLC） - 激活肌球蛋白II收缩功能的调节亚单位--富含于 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 拆卸，分别。这些结果暗示收缩性NMII是AIS的一种新的调节剂，并提示 一个保守的作用，在节点上，一个概念强烈支持MLC敲除研究。我们还发现 就在髓鞘形成之前，神经胶质细胞通过网格蛋白介导的细胞因子， 胞吞作用（CME）。我们的研究结果表明，清除的蛋白质不与细胞骨架连接，因此可以 聚集用于内吞作用。与经工程化以结合至NF 186的突变体NF 186构建体的表达一致， 结间细胞骨架，不被内吞，而是沿轴突沿着持续表达。引人注目的是，这 构建延迟髓鞘形成。后一项发现表明，间隙在塑造节点方面具有双重作用， 准备轴突用于髓鞘形成，从而协调朗维尔结的组装和 轴突在这里，我们测试了这个模型的关键方面，包括MLC/肌球蛋白II在节点组装/稳定性中的作用。 和肌动蛋白环的完整性。我们亦会研究有关机制， 这种神经胶质驱动的轴突蛋白清除的后果，包括在小鼠中建立有缺陷的CME模型， 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 重要的新见解轴神经胶质细胞的相互作用，调节节点的形成和髓鞘，潜在的， 导致有髓神经纤维紊乱的发病机制。