Worm Breeder's Gazette 13(5): 40 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
A protein fusion between the synaptic vesicle associated membrane protein VAMP and GFP localizes relatively specifically to synaptic vesicles permitting the visualization of synaptic terminals in live animals. VAMP (also called synaptobrevin and formally dubbed snb-l ) is a member of a family of proteins that have been implicated in vesicular docking and fusion. VAMP is a small protein of 120 amino acids with its C-terminus anchored into the synaptic vesicle membrane (fig lA). Antibodies directed against C. elegans VAMP co-localize with another synaptic vesicle associated protein, synaptotagmin. I chose to fuse GFP to the C-terminus of VAMP because R. Kelly's lab (UCSF) had demonstrated that sequences sufficient to localize VAMP to synaptic vesicles were present in the cytoplasmic domain of VAMP. The construct inserts the GFP coding sequence in frame just prior to the termination codon in a snb-l genomic clone. The resulting fusion protein would be predicted to be oriented in the synaptic vesicle membrane as illustrated in figure lB. When the VAMP-GFP construct is present in nematodes as an integrated array, called jsIsl, synaptic rich regions of the nervous system can be visualized directly using epi-fluorescence. The ventral cord, dorsal cord, nerve ring, and portions of the pharyngeal nervous system fluoresce brightly. In most animals commissures are not visible, nor are the dendritic portions of the amphid bundles. The cell bodies of some neurons are detectable. In addition, discrete patches of fluorescence in sub-lateral bundles and the SAB processes are readily visible in all animals. Finally, in young adults patches are also visible in the proper location in the vulval region to represent the terminals of HSN and VC neuron synapses with vulval muscles. Unfortunately, in jsIsl adults GFP-derived fluorescence appears in the spermatheca and proceeds to spread through the central part of gonad in older adults making the visualization of these patches difficult. While it is unlikely that all of the neuronal VAMP-GFP fusion product is localized to synaptic vesicles, several lines of experiment suggest that most of the fusion product is vesicularly localized. First, in unc-104 (el265); jsIsl animals fluorescence is concentrated in neuronal cell bodies and absent from the dorsal cord. This suggest that most axonal membrane does not contain significant amounts of VAMP-GFP. Second, in most jslsl; unc 5 (e53) animals variable sized patches of fluorescence are observed in sub-lateral and lateral positions. These patches are likely to be the ectopic neuromuscular synapses which Hedgecock, Culloti and Hall (1990) observed in unc-5 (e53) animals. Third, in strains bearing extrachromosomal arrays expressing the VAMP-GFP fusion under control of the mec-7 touch cell promoter, fluorescence in the nerve ring was limited to several patches ventrally and laterally. These patches were in similar positions as the synaptic connections described for the mec-7 expressing neurons ALMR, ALML and AVM in White et al's 'the mind of a worm'. Finally, in collaboration with Yishi Jin in Bob Horvitz's lab VAMP-GFP was placed under the unc-25 (glutamic acid decarboxylase) promoter. These VAMP-GFP constructs yielded a patchy fluorescent pattern in the ventral and dorsal cord and some nerve ring staining. The numbers of patches in the cords are consistent with the number of GABAergic inhibitory neuromuscular synapses formed by GABAergic neurons (personal communication, E. Jorgensen). Taken together these data provide strong evidence that the VAMP-GFP fusion localizes to synaptic vesicles when expressed in C. elegans. I am presently using VAMP-GFP fusions to screen for mutants with synaptogenesis defects. My work with VAMP and VAMP-GFP was initiated while I was a postdoctoral fellow in Dr. Barbara Meyer's laboratory.