Role of Gap Junctions in the Regulation of Cell Migration

该项目结合了识别间隙连接和测量细胞迁移的尖端技术。我们将研究几种类型的肾上腺细胞，它们在间隙连接表达方面表现出广泛的变化，这要么是因为细胞的初始表型，要么是因为细胞内第二信使的实验性刺激/抑制。在这些研究中，表面间隙连接改变对细胞迁移调控的影响将被深入探讨。间隙连接在细胞迁移中起关键作用的假设将被解决。迁移将在具有或不具有间隙连接的肾上腺细胞培养中进行研究（特异性目的1）。细胞将用特定的cAMP拮抗剂、激动剂或稀释剂处理，并评估其迁移特性。迁移将使用以下技术进行分析：(1)用膜透性染料预先标记的细胞的核位移或细胞前缘位移的延时成像；(2)计算从创面边缘向剥落区域移动的细胞数量；(3)计算在硅模板区隔室、微流控室或通过博伊登室过滤器迁移的细胞数量。延时视频显微镜和免疫细胞化学技术的结合将用于识别和阐明间隙连接动力学在细胞迁移中的作用。基础设施贡献/更广泛的影响该实验室将继续为培养本科生和研究生、博士后、少数民族院校的客座教授以及指导高中生和（影子计划）教师提供资源。过去的NSF研究基金为细胞生物学、内分泌学和信号转导领域的学生、博士后研究员和教师提供了机会和培训。通过在国内和国际上举办的研讨会和讲习班，已经并将继续向科学界成员提供来自若干学科的信息。高中生将继续发展研究项目，并在市和州的科学博览会上展示。最近，两名本科生在全国会议上获得了第一名，其他人将被鼓励走上他们的道路。

This project incorporates cutting-edge techniques for the identification of gap junctions and measurements of cell migration. Several types of adrenal cells will be studied that exhibit a wide variation in gap junction expression, either because of the initial phenotype of the cell or through experimental stimulation/inhibition of second messengers within the cell. In these studies, the influence of altered surface gap junctions on the regulation of cell migration will be explored in depth. The hypothesis that gap junctions play a pivotal role in cell migration will be addressed. Migration will be studied in cultures of adrenal cells that either have or do not have gap junctions (Specific Aim I). Cells will be treated with specific cAMP antagonists, agonist or diluent, and their migration characteristics will be assessed. Migration will be assayed using the following techniques: (1) time lapse imaging of the nuclear displacement or cell leading edge displacement of cells prelabeled with membrane permeable dyes; (2) counting the number of cells moving from a wound edge into a denuded area; and (3) counting the number of cells migrating in a silicon template compartmentalization chamber, in a microfluidic chamber, or through a Boyden chamber filter. A combination of time-lapse video microscopic and immunocytochemical techniques will be used to identify and elucidate the role of gap junction dynamics in cell migration.Infrastructural Contributions/Broader ImpactThe laboratory will continue to serve as a resource for the training of undergraduate and graduate students, postdoctoral fellows, visiting professors from minority institutions, and for mentoring high school students and (shadow program) teachers. Past NSF research funding has provided for the exposure and training of students, postdoctoral fellows, and faculty in areas of cell biology, endocrinology and signal transduction. Through seminars and workshops presented both nationally and internationally, information has been, and will continue to be, provided to members of the scientific community from a number of disciplines. High school students will continue to develop research projects and present at city and state science fairs. Recently, two undergraduate students won first place awards at national meetings, and others will be encouraged to follow in their path.