Capacitance &Fluorescence Monitoring of Exo/Endocytosis

University Of Colorado Denver, Aurora CO

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the mechanisms by which secretory granules in neuroendocrine cells undergo exocytosis and are retrieved subsequently by endocytosis. We will use a combination of electrophysiological (cell-attached and whole cell patch clamp capacitance) and optical (FM dye fluorescence) techniques to examine single exocytic events in rat pituitary lactotrophs. We showed previously that the secretory behavior of lactotrophs makes them particularly advantageous for such studies. In these cells, it is possible to observe single exocytic events in real-time with FM dyes using an ordinary epifluorescence microscope. We showed that fusion pores in these cells explode open upon stimulation, that granule-granule fusion (compound exocytosis) is a common phenomenon and is regulated independently of granule-plasma membrane fusion, and that the solubilization and release of dense cores is a regulated process. We propose to continue these studies. In preliminary experiments using cell-attached capacitance recordings, we have identified an additional, altogether new type of exocytic behavior, namely spontaneous, rhythmic, pulsatile openings of fusion pores. The phenomenon is common (evident in nearly 1/4 of patches) and robust (persisting unaltered for tens of minutes). The individual fusion pore openings are remarkably regular in time (frequency about 1 Hz) and the duration of the fusion pore openings is remarkably constant (ca. 50 ms). We propose to examine this behavior, its underlying mechanisms, and the cellular processes that regulate it in the next grant period. Implications for human health. All cells engage in exocytic secretion, a vital and complex process that is only partly understood, and which, if disrupted, can lead to impaired cellular function and ill health. While our work focuses on this basic cellular process, and not a specific disease entity, a better understanding of the fundamental mechanisms that regulate these vital functions could significantly impact our ability to combat a variety of diseases.