Melatonin Mediated Reorganization of the Cytoskeleton

Melan, Melissa

Abstract

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.