Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.Proper functioning of the nervous system requires fast, spatially-restricted neuron- neuron communication at synapses. Classic physiology studies have demonstrated the importance of calcium in regulating synaptic communication; however the molecular events underlying basic synaptic transmission and plasticity have only recently become the subject of intense investigation in neuroscience. The synaptotagmin family of vesicle proteins has been implicated in calcium- dependent neurotransmitter release, although Synaptotagmin 1 (Syt 1) is the only isoform demonstrated to control synaptic vesicle fusion. We have characterized the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologs of mammalian Synaptotagmins 4, 7, 12 and 14. Using immunolocalization and in situ hybridization experiments (Chapter 2), we demonstrate that each isoform has a unique subcellular localization and expression pattern, suggesting only Synaptotagmin 1 functions in synaptic vesicle exocytosis. Consistent with their distinct localizations, overexpression of Synaptotagmin 4 (Syt 4) or Synaptotagmin 7 (Syt 7) cannot functionally substitute for the loss of Syt 1 in synaptic transmission and loss-of-function mutations in Syts 4 and 7 do not have defects in neurotransmitter release (Chapter 4). Rather, Syt 4 and Syt 7 likely function in novel regulated-exocytosis pathways within neurons, distinct from synaptic vesicle cycling. The unique ability of Syt 1, but not other Syt isoforms, to localize to synaptic vesicles prompted us to determine the domains within Syt 1 responsible for its trafficking to synaptic vesicles (Chapter 3). We find the trafficking of Syt 1 is complex, likely requiring several sorting signals present at the N-terminus and in the C2 domains.(cont.) The N-terminus was required for proper targeting to presynaptic terminals, while the C2 domains were essential for internalization at synapses. Furthermore, we demonstrate the C2 domains of Syts 4,7, [alpha] and [beta] can not promote localization to synaptic vesicles, even if mislocalized to presynaptic terminals, further arguing only Syt 1 is present on synaptic vesicles in vivo (Chapter 3). Like Synaptotagmin 1, Syt 4 is ubiquitously present at most, if not all synapses, but localizes to the postsynaptic compartment (Chapter 2). Syt 4 homologs have been identified in all metazoan genomes sequenced to date, suggesting this isoform may mediate an evolutionarily conserved role in postsynaptic vesicle trafficking. To elucidate the function of Syt 4-dependent postsynaptic vesicle trafficking we have generated and analyzed null mutations in syt 4. Although Syt 4 is not required for viability, embryonic neuromuscular junctions in mutant animals show a developmental delay in the formation of varicosities, a reduction in neurotransmitter release, and loss of a particular form of synaptic plasticity following high frequency stimulation, we have termed HFMR (High Frequency-induced Miniature Release). Postsynaptic expression of a syt4 transgene can rescue the presynaptic defects (Chapter 4), indicating Syt 4 mediates a retrograde signaling pathway at synapses. In addition, we demonstrate Syt 4 cycles through the postsynaptic plasma membrane (Chapter 4), suggesting it may regulate secretion of retrograde signals in a manner analogous to Syt 1 regulation of neurotransmitter release, presynaptically. There is mounting evidence in several experimental systems for a regulated form of postsynaptic vesicular trafficking.(cont.) Dendritic release of a number of neuromodulators such as dopamine, ATP, GABA, and neuropeptides has been documented, while postsynaptic vesicles within dendritic spines and shafts have been directly visualized using electron microscopy. Studies in hippocampal culture neurons indicate that long-term labeling with FMI-43 loads dendritic organelles that undergo rapid calcium-triggered exocytosis. The localization and evolutionary conservation of Syt 4 makes it an attractive candidate for mediating a postsynaptic trafficking pathway common to all metazoan synapses. Indeed, localization studies of Syt 4 in mammals have noted the presence of the protein within dendrites and soma, similar to our studies in Drosophila. Utilizing the exceptional genetic tools available to Drosophila, we expect the characterization of Syt 4 and this novel retrograde signaling pathway will lead to new and exciting insights into the role of this protein family in fundamental synapse biology.by William W. Adolfsen.Ph.D
