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 Impact
The 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.
