Genetic study of gap junction formation and regulation in C. elegans neurons

缝隙连接在许多生物过程中起着重要的作用，如胚胎发育、细胞 分化、细胞生长、无血管器官的代谢协调和神经发育，以及 对缝隙连接的错误调控与许多疾病有关。然而，分子机制 潜在的缝隙连接的形成和调控在很大程度上仍然是未知的。利用线虫PLM神经元作为研究对象 模型中，我们发现功能性的GFP标记的内联蛋白形成了代表缝隙位置的斑块结构 活体内的连接。然后，我们使用表达GFP标记的转基因进行了无偏见的遗传筛查 发现12个突变体，在缝隙连接中存在3种类型的缺陷。根据从中国分离的突变体 在这一遗传筛选中，我们概述了3个目标，即研究缝隙连接形成的机制， 换人和淘汰。在初步研究中，我们发现了CED先前未知的功能-- 10/RAC和MEC-15/Fbox/WD重复蛋白在调控缝隙连接形成中的作用 目的1研究CED-10和MEC-15在缝隙连接中的功能和调控 队形。缝隙连接翻转的调节在缝隙连接功能中起着重要作用。在我们之前的 研究发现，线虫锚定蛋白UNC-44和CRMP UNC-33调节缝隙连接 营业额。然而，无论是UNC-44还是UNC-33突变体都没有完全穿透的表型，这表明 其他途径也参与调节缝隙连接的周转。在这项提案中，我们提出证据以表明 线虫Titin UNC-22的功能与UNC-44/UNC-33途径平行，可能受到调控 通过微管调节缝隙连接的周转。在目标2中，我们建议研究的调节机制 UNC-22/Titin和微管及其在缝隙连接周转中与UNC-44/UNC-33通路的串扰。 在神经元发育过程中，缝隙连接通道在缝隙连接前的细胞膜上表达 在此阶段，人们对缝隙连接通道的分布和功能知之甚少。我们 结果表明，在缝隙连接形成之前，UNC-9/innexin在PLM神经元中沿轴突形成点状结构。 而这些瞬时的缝隙连接通道簇被自噬途径消除，当神经元 形成缝隙连接。在目标3中，我们概述了一项计划，以解决这些间隙连接的瞬时簇的功能 调节神经元发育的通道和自噬途径。完成这项提案将导致 对于发现缝隙连接形成和调控的新机制，线虫的建立 PLM神经元作为研究缝隙连接的强大模型，为进一步研究提供了新的工具。 鉴于许多神经障碍与缝隙连接缺陷有关，这个项目可能会帮助 了解大脑在生理和病理条件下的发育和功能。

Gap junctions play essential roles in many biological processes, such as embryo development, cell differentiation, cell growth, metabolic coordination of avascular organs, and neural development, and misregulation of gap junctions has been linked to many diseases. However, the molecular mechanisms underlying gap junction formation and regulation are still largely unknown. Using C. elegans PLM neurons as a model, we found that functional GFP-tagged innexins form plaque structures that represent the location of gap junctions in vivo. We then carried out an unbiased genetic screen using transgenes expressing GFP-tagged innexins and uncovered 12 mutants with 3 types of defects in gap junctions. Based on mutants isolated from this genetic screen, we outline 3 aims in this proposal to study mechanisms for gap junction formation, turnover, and elimination. In the preliminary studies we discovered the previously unknown function of CED- 10/Rac and MEC-15/ F-box/WD repeat-containing protein in regulating gap junction formation, and in Specific Aim 1 we outline a plan to investigate the function and regulation of CED-10 and MEC-15 in gap junction formation. Regulation of gap junction turnover plays an important role in gap junction functions. In our previous study, we revealed that the C. elegans ankyrin protein UNC-44 and CRMP UNC-33 regulate gap junction turnover. However, neither unc-44 nor unc-33 mutants have completely penetrant phenotypes, suggesting that other pathways are involved in regulating gap junction turnover. In this proposal we present evidence to show that the C. elegans titin UNC-22 functions in parallel with the UNC-44/UNC-33 pathway and is likely regulated by microtubules to modulate gap junction turnover. In Aim 2 we propose to study the regulatory mechanisms of UNC-22/titin and microtubules and their crosstalk with the UNC-44/UNC-33 pathway in gap junction turnover. During neuronal development gap junction channels are expressed on the membrane before gap junction formation, and little is known about the distribution and function of gap junction channels at this stage. We show that UNC-9/innexin forms puncta along the axon in PLM neurons before the formation of gap junctions, and these transient clusters of gap junction channels are eliminated by the autophagy pathway when neurons form gap junctions. In Aim 3 we outline a plan to address the function of these transient clusters of gap junction channels and the autophagy pathway in regulating neuronal development. Completion of this proposal will lead to the discovery of novel mechanisms of gap junction formation and regulation, the establishment of C. elegans PLM neurons as a powerful model to study gap junctions, and the generation of new tools for further studies. Given that many neural disorders are associated with defects in gap junctions, this project will likely aid in the understanding of brain development and functions in both physiological and pathological conditions.