Mechanisms of Node of Ranvier Assembly

轴突起始段(AIS)和兰维尔结节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。它们在生电中的关键作用 由电压门控钠(NAV)和钾组成的大分子络合物的显著富集性 (KCNQ)通道、细胞黏附分子(NF186、NrCAM)和AnkG细胞骨架支架(AnkG) 和β-IV(βIV)血影蛋白。在髓鞘形成之前，结节成分沿着轴突弥漫分布。 与其持续的动作电位传导相一致。随着髓鞘的形成，轴突重组为 离散的结构域，在节点组装中达到顶峰，从而实现跳跃传导。一个关键的问题是 推动了这场重组？我们发现两个相辅相成的机制在很大程度上有助于：一)招聘 在节处靶向和稳定该复合体的信号以及ii)从结节上去除结蛋白的主动清除 沿着轴突的其他地方。在这里，我们研究这两种机制对节点形成的贡献。 三叉神经节和中枢神经系统节点的招募信号最终组装成AnkG/βIV光谱蛋白细胞骨架支架 所有其他节点组件都绑定到该节点。这个脚手架被进一步捆绑在一起，并可能通过定期的 AIS和结节均有间隔的亚膜肌动蛋白环。我们最近在AIS报告了肌动蛋白环 收缩肌球蛋白II特异性地修饰结节。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-富含在肌球蛋白II的早期标志物 AIS和节点。增加或降低pMLC水平/肌球蛋白II活性的策略，推动AIS组装和 分别进行了反汇编。这些结果表明，收缩NMII是AIS的一种新的调节因子，并提示 节点处的保守角色，这一概念得到了MLC基因敲除研究的大力支持。我们还发现， 就在髓鞘形成之前，神经胶质细胞通过网状蛋白介导的方式驱动节间成分的清除。 内吞作用(CME)。我们的结果表明，清除的蛋白质与细胞骨架没有联系，因此可以 聚集在一起进行内吞作用。一致表达突变型NF186结构，该结构经工程处理后与 节间细胞骨架不是内吞的，而是沿着轴突持续表达。令人惊讶的是，这 构建延缓了髓鞘形成。后一项发现表明，间隙在雕刻结点和 为髓鞘形成的轴突做准备，从而协调Ranvier结节与髓鞘形成的组装 轴突。在这里，我们测试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 和肌动蛋白环通过击倒和击倒策略的完整性。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 对调控结节形成和髓鞘形成的轴突-神经胶质相互作用的重要新见解，以及潜在的， 研究导致有髓纤维紊乱的致病机制。

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.