Assembly of the Node of Ranvier

描述(申请人提供)：动作电位产生于轴突起始段(AIS)，并通过跳跃传导在Ranvier结点传播。这些结构域在电压门控的Na+通道中高度丰富，与β亚基、神经细胞黏附分子，特别是神经法菌素(NeuroFasin，NF)186，以及细胞骨架蛋白ankyrin G和PIV Spectrin形成多聚体复合体。我们最近证明，NF186靶向PNS结节是通过其细胞外相互作用介导的，它通过细胞质结构域招募Ankyrin G在结节形成中发挥重要作用，而Ankyrin G反过来又是钠通道积累所必需的。相反，在初始片段的锚蛋白积累，这对结构域组织也是至关重要的，是内在地指定的，独立于NF186。这些研究提出了一些关键问题。针对该节点的蛋白质的来源是什么？它们是如何传递到该站点的？结节组件一旦组装，是否在结节处稳定表达，或者它们是否持续周转？如果是，这种周转是否因脱髓鞘而增强？最后，CNS节点是像PNS节点一样组装(即由细胞外信号引导)还是更类似于初始片段(即通过与ankyrin G的相互作用由内而外引导)？在这个建议中，我们解决这些问题，并通过以下方式进一步描述节点组装的机制：i)确定蛋白质如何运输到PNS节点，包括它们是从细胞表面池招募的还是通过直接的囊泡运输；ii)活图像节点以检查在发育和脱髓鞘过程中发生的动态变化；以及iii)研究CNS节点对共培养中依赖NF186的信号和转基因小鼠中靶向信号的依赖。这些研究应该为调控Ranvier结节组装和维护的轴突-神经胶质相互作用提供重要的新见解。它们也将是阐明脱髓鞘导致的结节病理性变化的重要基础。

DESCRIPTION (provided by applicant): Action potentials are generated at the axon initial segment (AIS) and are propagated via saltatory conduction at nodes of Ranvier. These domains are highly enriched in voltage gated Na+ channels, which form a multimeric complex with beta subunits, neuronal cell adhesion molecules, notably neurofascin (NF) 186, and the cytoskeletal proteins ankyrin G and piv spectrin. We have recently demonstrated that targeting of NF186 to PNS nodes is mediated via its extracellular interactions, that it has an essential role in node formation by recruiting ankyrin G via its cytoplasmic domain, and that ankyrin G in turn, is required for the accumulation of sodium channels. In contrast, ankyrin accumulation at initial segments, which is also critical for domain organization is intrinsically specified, independent of NF186. These studies raise a number of key questions. What is the source of proteins targeted to the node and how do they traffic to this site? Are nodal components, once assembled, stably expressed at the node or do they continuously turnover and, if so, is this turnover enhanced by demyelination? Finally, do CNS nodes assemble like PNS nodes (i.e. directed by extracellular signals) or more akin to initial segments (i.e. directed from the inside-out via interactions with ankyrin G)? In this proposal, we address these questions and further characterize mechanisms of node assembly by: i) determining how proteins traffic to PNS nodes, including whether they are recruited from cell surface pools or via directed vesicular transport, ii) live image nodes to examine dynamic changes that occur during development and with demyelination, and iii) examine the dependence of CNS nodes on NF186- dependent signals in cocultures and targeting signals in transgenic mice. These studies should provide important new insights into the axo-glial interactions that regulate the assembly and maintenance of nodes of Ranvier. They will also be an important foundation for elucidating pathogenetic changes at nodes that result from demyelination.