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，Melan博士的长期目标是了解 功能性细胞骨架变化取决于 微管和微丝系统，例如在神经元中发现的微管系统 细胞。编码微管的细胞骨架基因的转录 微丝及其相关蛋白在 神经元的发展。褪黑激素的一个特性是其设施 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蛋白信号通路作用。两种类型的褪黑激素 ML1受体ML1A和ML1B显示出组织特异性分布，表明 它们具有不同的功能角色。转变的中国仓鼠卵巢 梅兰博士在现场合作者，博士博士提供了单元（CHO）线路。 witt-enderby，表达ML1A或ML1B受体。在 梅兰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经历细胞骨架重排 最终形成了长丝状产物 神经突的特征。此外，微管对于 这些细胞骨架变化发生。电流中的工作假设 提议是通过ML1A受体作用的褪黑激素激活A 启动通常在 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，计划了 褪黑激素刺激的CHO细胞和神经生长因子或褪黑激素 - 刺激的神经母细胞瘤细胞。在AIM 1中，Melan博士将确定 CHO和神经母细胞瘤细胞的细胞体结构变化的时间过程 与她的合作者Lanni博士在卡内基·梅隆 研究所。在刺激后各个阶段对细胞的北部分析 将提供有关是否表达特定细胞骨架的信息 基因与细胞结构变化相关。一代 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聚。在 AIM 2，Melan博士将评估微型若丹明标签的命运 肌动蛋白在褪黑激素治疗过程中随时间流逝显微镜检查 细胞。肌动蛋白解聚剂（例如细胞切拉蛋白D）的作用， 预计会增加产物的频率，而腓-phalloidin（a） 肌动蛋白分解的抑制剂可能会减少产物 抑制肌动蛋白分解可能会减少产物。 NGF必须 在神经元细胞系中形成神经突在中 体外。最近，褪黑激素已被证明会诱导神经突样生长 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AIM 3中，需要在 维持CHO细胞和神经母细胞瘤细胞的产物将 通过相对比显微镜监测。应该没有生长 在没有褪黑激素的情况下缩回 神经丝将通过免疫荧光评估。在AIM 4中 抑制剂和激活剂对推定信号通路的影响 将在褪黑激素诱导的CHO细胞中确定。数字和 细胞的形态计量学将在每个遵循微观测量 治疗。