The goal of this project is to systematically study the structural underpinnings of exocytosis and endocytosis in a relevant and tractable cell-culture system that properly reflects the special properties of neurons and neuroendocrine cells. This reflects the applicant's long-term interest in elucidating the mechanisms of membrane recycling that link exo-and endocytosis at the synapse, where these mechanisms are fundamental to neuronal communication, learning, and memory. The applicant has chosen to study PC12 cells (derived from an immortalized rat pheochromocytoma) because they have been well-characterized biochemically and physiologically, they have a known complement of neuronal-type proteins involved in both secretion and endocytosis, and they have been used extensively in the field, to date. The applicant's specific approach will be to apply the special technique of """"""""deep-etch"""""""" electron microscopy (EM) pioneered in his laboratory to obtain 3-D images of structural changes that occur in PC12 cells attached to adhesive substrates and """"""""unroofed"""""""" with an ultrasonic jet of buffer to expose their inner membrane surfaces. That is where all the exo-and endocytotic events occur, and where the applicant intends to witness membrane changes and identify their molecular underpinnings. To accomplish this goal, the applicant will use his lab's new developments in digital/computer-based techniques for creating """"""""anaglyph"""""""" 3-D images of """"""""deep-etch"""""""" EM's, combined with novel methods of identifying molecules and organelles by :1) EM immunocytochemistry with gold-labeled secondary antibodies and 2) transiently expressed epitope-tagged molecules that can either be seen directly by """"""""deep etch"""""""" EM, or secondarily after DAB-based histochemistry is performed on them. This will permit the applicant to establish a close correlation between the """"""""static"""""""" images of PC12 cells obtained by EM and the """"""""dynamic"""""""" images of PC12 cells they plan to obtain by real-time confocal light microscopy. By this combination of powerful and modern microscopic imaging techniques, the applicant hopes to deepen our understanding of the basic cellular mechanisms that mediate neuronal communication, which should help to advance neuropharmacology and therapeutics and begin to address some of the fundamental questions about what goes wrong with synaptic communication in neuronal degenerative diseases such as Alzheimer's.
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