1987-1993: Synaptic Vesicles and the Nature of Thinking
I first became interested in synapses and synaptic vesicles during my doctoral research at Stanford. On day one of my graduate school training, my advisor, Richard Scheller, assigned me the task of purifying synaptic vesicles (SVs) from the electric organs of electric rays. The stated purpose of this was, as he put it, to identify the “proteins that mediate thinking”. Although I found that description of the project humorous, I quickly saw the truth in it. I soon realized that this was a research area poised for a revolution. The basic idea was to purify SVs from a tissue where they were highly enriched and resolve the proteins using polyacrylamide gel electrophoresis. We would then raise antibodies against individual proteins or try to sequence fragments of the protein for the purpose of screening cDNA libraries to identify the proteins that promote the fusion of SVs with the plasma membrane. No such proteins had been identified when we started the project. Synaptic Vesicles from a High Voltage Tissue Richard chose electric organs because they have a high density of SVs and all the SVs in this tissue secrete the same neurotransmitter (acetylcholine). The synchronous release of billions of SVs in this organ generates a high voltage that can stun/ paralyze prey. It can also cause a painful shock to humans (I'm speaking from personal experience!). I installed tanks in the lab and periodically ordered shipments of 30 or so rays from a company that got them from fishermen in Mexico. I would feed and care for them until "dissection day", on which I recruited all lab members to help anesthetize the animals and do the dissections. The prep started with a series of centrifugal fractionations and a sucrose gradient. We followed the SVs with ATP using a firefly luciferase assay. In the final step, the peak fractions from the sucrose gradient were loaded on a 7 ft CPG column (Controlled Pore Glass). This fractionated the small uniform SVs away from larger contaminating irregular membrane fragments. I struggled for over a year to get the prep to work and almost gave up and quit graduate school over it. At one point I drove up to Regis Kelly's lab at UCSF, where the prep was developed, and his technician kindly demonstrated it for me. I'm not sure what made the difference, but at some point it started to work. G proteins on Synaptic Vesicles??? Shortly after the prep was working, a postdoc in the lab, Johnny Ngsee, used it for a very interesting experiment. He ran the CPG column fractions on a gel and blotted them to nitrocellulose. He then overlaid the blot with 32P-GTP to determine if any GTP-binding proteins co-purified with the vesicles. Amazingly, the experiment worked! He found both low molecular weight G proteins (now known as Rabs) and at least one high molecular weight G protein from the pertussis toxin sensitive class (now known as G alpha i or G alpha o). Later, when I switched to C. elegans, we and others found that G alpha o has strong effects on neurotransmitter release and behavior (Adventures>1996-1999) Ngsee JK, Miller K, Wendland B, and Scheller RH (1990). Multiple GTP-Binding proteins from cholinergic synaptic Vesicles. J Neurosci. 10, 317-322. PMID: 2105379. Synaptotagmin!
It soon became clear that this first-ever proteomic study of SV proteins was more than a single grad student could handle, especially when we knew we had competitors who were after the same goal. Richard took on another graduate student, Beverly Wendland, to work with me on the SV project. He also purchased an Edman-Degradation protein sequencer and ultimately hired Jim Schilling, luring him away from his company job to work full-time on obtaining protein sequences from the gel bands that Beverly and I generated. Jim obtained a short stretch of sequence from one of the vesicle-specific proteins. About 2 weeks later, our main competitor Thomas Sudhof published a Nature paper reporting rat Synaptotagmin that was clearly related to the protein sequence we had. Beverly used the protein sequence and the published rat Synaptotagmin sequence to design oligos and screened an electric organ cDNA library. She pulled out clones for 3 different isoforms (related proteins) predicted to encode proteins of 62 - 74 kDa. We called them A, B, and C. We then produced or obtained antibodies specific for each of the 3 electric ray Synaptotagmin isoforms. Using Western Blotting, I demonstrated that only the B isoform was present in electric organ, and it precisely co-purified with the SV fractions of the CPG column. The antibody also recognized some higher molecular weight forms of the protein. The 2 highest forms were non-reduced species due to our DTT being old. I undertook a project to sort out where each isoform was expressed in various regions of the electric ray nervous system. We didn't have good antibodies to isoform C, so I focused on A and B. I taught myself some brain anatomy and learned how to preserve various brain and endocrine system parts for visualization with antibodies. Our lab neighbor Sue McConnell generously taught me how to cut thin sections of frozen formaldehyde-fixed tissue, stain them with our fluorescent-labeled antibodies, and use a high-power light microscope. I found that only the Synaptotagmin B isoform was localized to neuromuscular junction synapses. This is the expected location for a synaptic vesicle protein. Throughout the nervous system and in many endocrine and gut tissues, I found that where B was highly expressed, A was weakly expressed or absent and vice versa. In general, the B isoform was expressed most highly in lower brain regions such as the brainstem and spinal cord, and the A isoform was expressed in higher brain regions such as the forebrain (including the cerebral cortex). Wendland B, Miller KG, Schilling J, and Scheller, RH (1991). Differential expression of the p65 gene family. Neuron 6, 993-1007. PMID: 2054189. [Footnote states first 2 authors contributed equally] cited by 101 papers as of April, 2018 |
Fun Factoids about this Study
Since the discovery of Synaptotagmin by our study and the paper from Tom Suhof's lab, there have been 2436 papers published with Synaptotagmin as a key word!
Taking the Next Step
As I finished up my graduate training, I was pretty sure that I wanted to continue working on synaptic vesicles and synapses as a postdoc and ultimately a lab head. I knew that one challenge for the field was to investigate the functions of the SV proteins that were being discovered. The best way to do that was to eliminate the gene or protein in an animal model and determine the consequences for SV function and synaptic transmission. One concern I had about this approach is that others hadn’t had much success with it. For example, Synaptophysin is a major synaptic vesicle protein, but deletion of the Synaptophysin gene in mice had virtually no effect at either the behaviorally level or the level of recording synaptic transmission events from synapses. The mouse knockouts of 2 other SV genes, Synapsin and Rab3 gave similar results (weak or no phenotypes). My other concern with this approach is that it provided no direct route for discovering new synaptic proteins, since it just focused of the functions of synaptic proteins that had already been discovered.
To read about the genetic approach I took as a postdoc, see Adventures > 1993-1996.
To read about the genetic approach I took as a postdoc, see Adventures > 1993-1996.