The proposed project examines endocytic vesicle processing in the vertebrate nervous system, using the neuromuscular synapse as a model. The single-fiber-thick nerve-muscle preparation used (snake transversus abdominis) is highly stereotyped and permits electrophysiological, light microscopic, and electron microscopic evidence to be compared, all at the same identified synapse. The fluorescent probe sulforhodamine 101 is utilized as a marker for recently endocytosed vesicles. The probe is less phototoxic and photobleachable than those previously used to investigate vesicle processing, thereby permitting the first use of diffraction-limited 3-D confocal microscopy to visualize vesicle clusters and other structures in living and fixed preparations. The project's goal is to determine how a synapse """"""""chooses"""""""" among several processing pathways which have been identified in preliminary experiments: endocytosis at transmitter release sites, possibly via reabsorbtion vesicles, versus clathrin-mediated endocytotic process located elsewhere; endocytosis following calcium-dependent release versus endocytosis following calcium-independent release; and endocytosis leading to reformation of transmitter-containing vesicles versus endocytosis leading to retrograde transport towards the cell body. Because the strength of a synapse depends on its ability to maintain a supply of transmitter, knowledge of how transmitter vesicles are processed is essential in understanding diseases, such as Parkinson's, depression, and Eaton-Lambert's in which alteration of synaptic strength is the primary pathology.
| Barbieri, M A; Gumusboga, A; Roberts, R L et al. (2001) Measurement of Rab5 protein kinase B/akt and regulation of ras-activated endocytosis. Methods Enzymol 329:145-56 |
