MELATONIN MEDIATED REORGANIZATION OF THE CYTOSKELETON

褪黑激素介导的细胞骨架重组

• 批准号: 2669116
• 负责人: MELISSA A MELAN
• 金额: $ 9.77万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-28 至 2001-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2669116
• 关键词: CHO cells; actins; biological signal transduction; cell components; cell growth regulation; cytogenetics; cytoskeleton; gene expression; hormone regulation /control mechanism; immunofluorescence technique; melatonin; microtubule associated protein; microtubules; neurofilament; neurotrophic factors; northern blottings; phase contrast microscopy; photomicrography; polymerization

DESCRIPTION: The PI, Dr. Melan's, long term goal is to understand the functional cytoskeletal changes that are dependent on the action of microtubule and micro-filament systems, such as those found in neuronal cells. Transcription of the cytoskeletal genes encoding microtubules, micro-filaments, and their associated proteins are essential during the development of neurons. One property of melatonin is its facility to direct cytoskeletal changes in pigment and neuroblastoma cells. Melatonin acts through the G protein signaling pathway. Two types of the melatonin ML1 receptor, ML1A and ML1b, show tissue specific distribution, suggesting they have different functional roles. Transformed Chinese hamster ovary cell (CHO) lines are available from Dr. Melan's on site collaborator, Dr. Witt-Enderby, that express either the ML1a or ML1b receptor. In preliminary studies, Dr. Melan, has shown that the ML1a but not the ML1b line, when treated with melatonin undergoes cytoskeletal rearrangements that culminate in the formation of long filamentous outgrowths characteristic of neurites. Furthermore, microtubules are essential for these cytoskeletal changes to occur. The working hypothesis in the current proposal is that melatonin acting through the ML1a receptor activates a set of genes that initiate the cytostructural changes normally found in neuronal cells. The current objective is to determine how melatonin treatment influences cell shape. To address this issue, comparisons are planned between melatonin-stimulated CHO cells and nerve growth factor or melatonin- stimulated neuroblastoma cells. In Aim 1, Dr. Melan will determine the time course of cytostructural changes in CHO and neuroblastoma cells by time lapse microscopy with her collaborator, Dr. Lanni, at Carnegie Mellon Institute. Norther analysis of cells at various stage post-stimulation will provide information on whether expression of specific cytoskeletal genes are correlated with the cytostructural changes. The generation of a growth cone is typical of migrating fibroblasts and neurites and requires the break down and subsequent repolymerization of actin in the cone. In Aim 2, Dr. Melan will assess the fate of microinjected rhodamine-labeled actin with time lapse microscopy during melatonin treatment of the CHO cells. The effects of actin depolymerizing agents, such as cytochalasin D, are predicted to increase the frequency of outgrowths while phalloidin, a suppressor of actin breakdown may reduce outgrowths while phalloidin, a suppressor actin breakdown may reduce outgrowths. NGF is required to maintain the neurite after their formation on neuronal cell lines in vitro. Recently, melatonin has been shown to induce neurite-like growth from neuroblastoma cells but the effects of subsequent melatonin removal are not known. In Aim 3, the need for continuous melatonin on the maintenance of outgrowths from CHO cells and from neuroblastoma cells will be monitored by phase contrast microscopy. Should there be no outgrowth retraction in the absence of melatonin, the stabilizing effects of the neurofilaments will be assessed by immunofluorescence. In aim 4, the effects of inhibitors and activators on the putative signaling pathway will be determined in melatonin-induced CHO cells. The number and morphometry of the cells will be measured microscopically following each treatment.

描述：PI（梅兰博士）的长期目标是了解 功能性细胞骨架的变化取决于 微管和微丝系统，例如在神经元中发现的系统 细胞。编码微管的细胞骨架基因的转录， 微丝及其相关蛋白质在 神经元的发育。褪黑激素的特性之一是它能够 色素和神经母细胞瘤细胞的直接细胞骨架变化。褪黑激素 通过 G 蛋白信号通路发挥作用。褪黑激素的两种类型 ML1 受体 ML1A 和 ML1b 显示组织特异性分布，表明 他们有不同的功能角色。改造的中国仓鼠卵巢 细胞 (CHO) 系可从 Melan 博士的现场合作者 Dr. Witt-Enderby，表达 ML1a 或 ML1b 受体。在 Melan 博士的初步研究表明，ML1a 而不是 ML1b 线，当用褪黑激素治疗时会发生细胞骨架重排 最终形成长丝状生长物 神经突的特征。此外，微管对于 这些细胞骨架的变化就会发生。当前的工作假设 建议认为褪黑激素通过 ML1a 受体发挥作用，激活 启动通常在细胞中发现的细胞结构变化的一组基因 神经元细胞。 当前的目标是确定褪黑激素治疗如何影响 细胞形状。为了解决这个问题，计划对 褪黑素刺激的 CHO 细胞和神经生长因子或褪黑素- 刺激神经母细胞瘤细胞。在目标 1 中，Melan 博士将确定 CHO 和神经母细胞瘤细胞的细胞结构变化的时间过程 与卡内基梅隆大学的合作者 Lanni 博士一起使用延时显微镜 研究所。刺激后不同阶段细胞的 Norther 分析 将提供有关特定细胞骨架是否表达的信息 基因与细胞结构的变化相关。一代人 生长锥是迁移成纤维细胞和神经突的典型特征，需要 锥体中肌动蛋白的分解和随后的重新聚合。在 目标 2，Melan 博士将评估显微注射罗丹明标记的命运 褪黑激素处理 CHO 期间用延时显微镜观察肌动蛋白 细胞。肌动蛋白解聚剂的作用，例如细胞松弛素 D， 预计会增加生长的频率，而鬼笔环肽（一种 肌动蛋白分解的抑制剂可能会减少生长，而鬼笔环肽（一种 抑制肌动蛋白的分解可能会减少生长。 NGF 需要 在神经元细胞系上形成后维持神经突 体外。最近，褪黑激素已被证明可以诱导神经突样生长 来自神经母细胞瘤细胞，但随后去除褪黑激素的影响 不知道。在目标 3 中，需要持续补充褪黑激素 维持 CHO 细胞和神经母细胞瘤细胞的生长将 通过相差显微镜监测。是否应该没有任何生长 在没有褪黑激素的情况下回缩，褪黑激素的稳定作用 神经丝将通过免疫荧光进行评估。在目标 4 中， 抑制剂和激活剂对假定信号通路的影响 将在褪黑激素诱导的 CHO 细胞中测定。数量和 每个细胞的形态测量将在显微镜下测量 治疗。