MELATONIN MEDIATED REORGANIZATION OF THE CYTOSKELETON

描述：PI，梅兰博士的长期目标是了解 功能细胞骨架的改变依赖于 微管和微丝系统，如神经元中发现的那些 细胞。编码微管的细胞骨架基因的转录， 微丝和它们的相关蛋白质在 神经元的发育。褪黑素的一个特性是它易于 色素和神经母细胞瘤细胞的直接细胞骨架改变。褪黑素 通过G蛋白信号通路发挥作用。褪黑素的两种类型 ML1受体ML1a和ML1b显示出组织特异性分布，提示 它们扮演着不同的职能角色。改造的中国仓鼠卵巢 细胞(CHO)线可从梅兰博士的现场合作者Dr。 Witt-Enderby，表达ML1a或ML1b受体。在……里面 梅兰博士的初步研究表明，ML1a而不是ML1b LINE，当用褪黑激素治疗时，经历细胞骨架重排 最终形成了长的丝状突起 具有神经突起的特征。此外，微管对于 这些细胞骨架的变化就会发生。当前的工作假说 有观点认为，褪黑素通过ML1a受体激活一个 一组启动细胞结构变化的基因，通常在 神经细胞。 目前的目标是确定褪黑素治疗如何影响 细胞形状。为了解决这个问题，计划在以下方面进行比较 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，梅兰博士将决定 CHO和神经母细胞瘤细胞结构变化的时程变化 与她的合作者兰尼博士在卡内基梅隆大学进行时间推移显微镜检查 研究所。刺激后不同阶段细胞的Norther分析 将提供关于特定细胞骨架表达的信息 基因与细胞结构的改变有关。一代一代的 生长锥是典型的迁移成纤维细胞和轴突，需要 肌动蛋白在锥体中的分解和随后的再聚合。在……里面 目的2，梅兰博士将评估显微注射罗丹明标记的命运 褪黑素治疗CHO过程中肌动蛋白的时间推移显微镜观察 细胞。肌动蛋白解聚剂，如细胞松弛素D， 预计会增加突起的频率，而鬼臼毒素，一种 肌动蛋白分解的抑制物可能会减少突起，而鬼臼毒素，一种 抑制者肌动蛋白的分解可能会减少突起。需要NGF来 在神经细胞系上形成轴突后保持其生长 体外培养。最近，褪黑素被证明可以诱导轴突样生长。 来自神经母细胞瘤细胞，但随后去除褪黑素的效果 都是未知的。在目标3中，需要在 维持CHO细胞和神经母细胞瘤细胞的突起将 通过相差显微镜进行监测。如果没有结果的话 在没有褪黑素的情况下，药物的稳定作用 神经细丝将通过免疫荧光进行评估。在目标4中， 抑制剂和激活剂对可能的信号通路的影响 将在褪黑素诱导的CHO细胞中被确定。数字和 细胞的形态测量将在显微镜下测量每一个 治疗。

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.