(Verbatim from Applicant's Description): Neurons and neuroendocrine cells release neurotransmitters/ hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. For many years, the focus of the P1's laboratory has been to understand the processes underlying the secretory cycle of dense-core-vesicles (DCVs) in the adrenal chromaffin (AC) cell system. The physiological and molecular basis of secretion has been studied extensively, but the nature of the endocytotic processes that are coupled to exocytosis is only partly understood. As such, an area of intense current interest is the mechanism of endocytosis in secretory cells. Two competing hypotheses: complete fusion of vesicular membrane with plasma membrane followed by retrieval in clathrin-coated vesicles and """"""""kiss-and-run"""""""" exocytosis where vesicles never completely fuse and are recovered intact from the membrane have been debated for the last 25 years. Using capacitance measurements we have characterized a novel form of endocytosis, termed """"""""rapid endocytosis"""""""" (RE; half time of seconds), that occurs immediately after exocytosis is transiently triggered in bovine calf AC cells. This process is clathrin-independent and may be part of the """"""""kiss-and-run"""""""" mechanism. Recently, new preliminary data suggest that another type of membrane retrieval which we call """"""""slow endocytosis"""""""" (SE; half time of several minutes) is activated by more sustained physiological stimulation and may be involved in recovering vesicular membrane after full fusion. The hypothesis we will investigate here is that RE and SE are mechanistically as well as kinetically distinct and we will probe key components of the regulatory machinery to prove this point. There are three specific aims: (I) Determine the relationship between RE and SE and determine the physiological conditions under which either process is predominant. We will analyze the kinetic and regulatory properties of SE and determine if is mediated by clathrin coated vesicles. (2) Determine the role of different dynamin isoforms in RE and SE. Calf AC cells express both dynamin-l and 2 and our hypothesis is that dynamin-l specifically regulates RE while dynamin-2 regulates the SE. We will also identify which domains of dynamin are critical for its function and test for differential involvement of dynamin-binding partners in the two different forms of endocytosis. (3) Determine the role of calcium, calmodulin and calcineurin in RE and SE. We know that all these modulators are involved in RE but their mode of action and whether they also participate in SE is not known. To accomplish these aims we will use a multidisciplinary approach that includes biochemical, molecular and single-cell electrophysiological methods in chromaffin cells. This project will enhance our understanding of vesicle retrieval that underlies vesicle recvclin2 processes in secretory cells.
