CAPACITANCE & FLUORESCENCE MONITORING OF EXO/ENDOCYTOSIS

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DESCRIPTION (from applicant's abstract) Exocytosis is one of the processes by which cells secrete substance to the external world. The complex physiological steps underlying the secretory cycle are only partly understood, but are of fundamental importance to all cells. Most early information about exo-and endocytosis came from studies of neuronal synapses. However, the patch clamp capacitance technique (Neher and Marty, 1982) shifted emphasis increasingly to isolated secretory cells that could be patch clamped, criteria that preclude most nerve terminals from such studies owing to their small size. Today, the prototypical cell for studying the physiology of secretion is the adrenal chromaffin cell; more is known about the mechanism of secretion in this cell than any other. The applicants propose to study exocytosis in chromaffin cells - particularly questions concerning exocytic 'hot spots' and 'kiss and run' exocytosis - using a technique that they developed - FM1-43 fluorescence - in combination with patch clamp capacitance and with electron microscopy. The technique of FM1-43 fluorescence has proven useful for studying exo- and endocytosis in neurons, and more recently, in chromaffin cells (Smith and Betz, 1996). Exocytosis can be monitored because FM1-43 fluorescence increases more than 300 times when it binds to a membrane. Thus, when a secretory granule undergoes exocytosis and its membrane binds FM1-43, the overall fluorescence of the cell increases. The applicants will optimize the spatial resolution of their measurements to detect single exocytic and endocytic sites. They will then address questions like: Are the sites distributed randomly over the surface of the chromaffin cell, or are they clustered in 'hot spots'? Does the distribution change with different amounts of stimulation? In addition to studying the spatial nature of exo- and endocytosis, they will also study the events that occur immediately after exocytosis. For example, in neurons synaptic vesicles collapse after they undergo exocytosis and become part of the cell surface membrane (they are retrieved by endocytosis later at remote sites). In chromaffin cells, recent evidence suggests that granules do not ordinarily collapse after exocytosis, but pinch back directly, a phenomenon known colloquially as 'kiss and run' exocytosis. The applicants have developed a method to monitor the post-exocytic fate of granules, and by injecting drugs, peptides, and other agents into a cell through a patch pipette, they will study the normal regulatory processes that determine the fate of a secretory granule.