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

秀丽隐杆线虫神经元间隙连接形成和调节的遗传学研究

• 批准号: 10187665
• 负责人: Dong Yan
• 金额: $ 35万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10187665
• 关键词: Address; Animal Model; Animals; Ankyrins; Area; Autophagocytosis; Axon; Biological Process; Biology; Birth; Brain; Caenorhabditis elegans; Cell Differentiation process; Cells; Chemical Dynamics; Chemical Synapse; Chimeric Proteins; Clinical Research; Data; Defect; Development; Disease; Electrical Synapse; Embryonic Development; Ensure; F Box Domain; Functional disorder; Gap Junctions; Generations; Genetic Screening; Genetic study; Individual; Interneurons; Invertebrates; Knowledge; Link; Location; Mammals; Mechanics; Membrane; Metabolic; Microtubules; Modeling; Molecular; Nematoda; Nervous system structure; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Neurophysiology - biologic function; Outcome; Pathologic; Pathway interactions; Phenotype; Physiological; Play; Proteins; Regulation; Role; Sensory; Signal Transduction; Structure; Synapses; Transgenes; Vertebrates; WD Repeat; base; cell growth; connectin; gain of function; gap junction channel; genetic analysis; in vivo; insight; loss of function; mutant; nervous system disorder; neural circuit; neurodevelopment; neuron development; new therapeutic target; novel; organ growth; relating to nervous system; tool

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.

缝隙连接在许多生物学过程中起着重要的作用，如胚胎发育、细胞增殖、细胞凋亡等。 分化、细胞生长、无血管器官的代谢协调和神经发育，以及 缝隙连接的失调与许多疾病有关。然而，分子机制 潜在的缝隙连接形成和调节仍然是未知的。利用C. elegans PLM神经元作为一种 模型中，我们发现功能性GFP标记的连接蛋白形成代表缺口位置的斑块结构 junctions in vivo.然后，我们使用表达GFP标记的转基因进行了无偏倚的遗传筛选。 结果发现了12个突变体，其中3种缺陷类型的突变体存在于缝隙连接中。基于从 这个遗传筛选，我们概述了3个目标，在这个建议，以研究间隙连接形成的机制， 周转和淘汰在初步研究中，我们发现了CED以前未知的功能- 10/Rac和MEC-15/ F-box/WD repeat蛋白在调节缝隙连接形成中的作用，以及在特异性 目的1：探讨CED-10和MEC-15在缝隙连接中的功能和调控 阵间隙连接转换的调节在间隙连接功能中起着重要的作用。在我们以前 研究结果表明，C.线虫锚蛋白<$44和CRMP <$33调节缝隙连接 周转然而，unc-44和unc-33突变体都没有完全的外显表型，这表明， 其它途径参与调节间隙连接转换。在这份提案中，我们提出的证据表明， C.线虫肌联蛋白<$22与<$44/<$33通路平行发挥功能， 通过微管调节缝隙连接的转换。在目标2中，我们建议研究 在间隙连接转换中，微管蛋白和微管与微管蛋白-22/肌联蛋白和微管与微管蛋白-44/微管蛋白-33通路的相互作用。 在神经元发育过程中，缝隙连接通道先于缝隙连接在细胞膜上表达 形成，并且在此阶段对间隙连接通道的分布和功能知之甚少。我们 显示了在间隙连接形成之前，在PLM神经元中，β-9/连接蛋白沿着轴突形成斑点， 这些间隙连接通道的瞬时簇被自噬途径消除， 形成缝隙连接。在目标3中，我们概述了一个计划，以解决这些短暂的间隙连接簇的功能 通道和自噬途径调节神经元发育。完成这项提案将导致 到缝隙连接形成和调节的新机制的发现，C. elegans PLM神经元作为研究缝隙连接的有力模型，并为进一步研究产生新的工具。 鉴于许多神经疾病与缝隙连接缺陷有关，该项目可能有助于 了解大脑发育和功能在生理和病理条件。