Cell membrane dynamics during exocytosis in anterior pituitary cells

Abstract

Exocytosis is a key biological process, which controls the neurotransmission and release of hormones from cells. In endocrine cells, hormones are packed into secretory vesicles, and released into the extra-cellular environment via a few hundred nanometre wide holes in the plasma membrane (called fusion pores or porosomes). The cell membrane dynamics responsible for the formation of the fusion pore, as well as its structure, remain scantly understood and are very complex since several different types of exocytosis mechanisms have been postulated with several potentially occurring simultaneously. This project deals with the study of the cell membrane dynamics during exocytosis in anterior pituitary cells, and more especially gonadotrophs. Gonadotrophs secrete the main hormone responsible for ovulation: luteinising hormone (LH). A better understanding of the physical LH release process would allow improved contraception methods and the ability to reduce the sterility problem. The first component of this project was to investigate the viability of the anterior pituitary cells in terms of their well-being, and their capacity to secrete hormone in response to stimulation. Gonadotrophin-releasing hormone was principally used as the secretagogue because it induced an important release of LH. Because this project deals with gonadotrophs in particular, and since the anterior pituitary gland is composed of six different types of cells, the necessity to isolate the gonadotrophs from the other cells became obvious. A technique based on immunolabelled magnetic nano-beads was thus tested for its ability to achieve gonadotroph isolation. Three complementary imaging techniques were chosen in order to realize a fine study of the dynamics of the exocytotic sites occurring during secretion. Living pituitary and separated cells were imaged with atomic force microscopy as well as cells that had been fixed in order to obtain better resolution observation. AFM, along with scanning and transmission electron microscopy, studies of the cells revealed two different exocytotic site configurations: simple single fusion pores and a complex association of pores dealing with a simple primary site combined with secondary attachments.