Assembly of the Node of Ranvier

描述（由申请人提供）：动作电位在轴突初始段（AIS）产生，并通过Ranvier节点的跳跃式传导传播。这些结构域在电压门控的Na+通道中高度富集，与β亚基、神经元细胞粘附分子（特别是神经束蛋白（NF） 186）和细胞骨架蛋白锚蛋白G和piv谱蛋白形成多聚体复合物。我们最近证明，NF186靶向PNS节点是通过其细胞外相互作用介导的，它通过细胞质结构域募集锚蛋白G在节点形成中起重要作用，而锚蛋白G反过来又是钠通道积累所必需的。相比之下，初始片段的锚蛋白积累对结构域的组织也是至关重要的，它是内在指定的，与NF186无关。这些研究提出了一些关键问题。靶向节点的蛋白质的来源是什么？它们是如何到达这个位点的？结成分一旦组装，是否稳定地在结表达，或者它们是否不断地转换，如果是这样，这种转换是否通过脱髓鞘增强？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（即通过与锚蛋白G的相互作用从内到外引导）？在本提案中，我们解决了这些问题，并通过以下方式进一步表征了节点组装机制：i)确定蛋白质如何运输到PNS节点，包括它们是通过细胞表面池还是定向囊泡运输招募的；ii)实时图像节点来检查发育过程中和脱髓鞘过程中发生的动态变化；iii)检查共培养中CNS节点对NF186依赖信号的依赖性以及转基因小鼠的靶向信号。这些研究应该为调节Ranvier淋巴结的组装和维持的轴胶质相互作用提供重要的新见解。它们也将是阐明脱髓鞘引起的淋巴结病理变化的重要基础。

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.