Jeremy Curtis and Dr. Dixon Woodbury, Physiology and Developmental Biology
The original intent of my project was to compare the fusion rates of synaptic vesicles to a syntaxin artificial membrane with and without SNAP-25. Recent research has shown that modified synaptic vesicles may fuse to a syntaxin-only bilayer . Prior to this, the major dogma of the SNARE-induced hypothesis of vesicle fusion has included SNAP-25 as a necessary component.
Within the last year some research has begun to elucidate alternate possible functions of SNAP-25. Since writing my ORCA grant proposal, my research in Dr. Woodbury’s lab has been slightly altered to explore and control for some of these possibilities.
Based on some of our research, it is thought that SNAP-25 may form channels in lipid membranes when it is allowed to aggregate by oxidation of its cysteine residues to form disulfide bridges. There are four possible cysteine oxidation sites, at least one of which is thought to be bound to a palmititic acid tail. The remaining cysteines may allow formation of the SNAP-25 multimers .
The discovery that SNAP-25 may cause channels is very problematic for the experiments that I proposed to do. The Nystatin-Ergosterol method I planned to use for measuring vesicle fusion requires use of the voltage clamp technique to measure current spikes that occur each time a vesicle fuses. The SNAP-25 was repeatedly observed to cause ion channels, greatly interfering with my ability to observe and count vesicle fusion events, since fusion is measured using electrical current. Current readings are given by both vesicle fusions and the hypothesized SNAP-25 multimer ion channels. The unexpected channels may mask fusion events rendering the measurements inaccurate and unreliable. That would make the results of my planned experiments too ambiguous to use.
To eliminate this ambiguity, my experiments had to be modified. The first step to do this was to establish good controls for my experiments. I hypothesized that if SNAP-25 multimers were causing the channels, then reducing the samples with beta-mercaptoethanol would eliminate them and I could proceed with my original experiments. The first step I took was to add SNAP-25 samples that had previously given channels, to the artificial membrane. After trying four samples, each at least three times (for a total of n=16 experiments) I saw no channels. Normally, this would have been good reason to proceed with my planned experiments, but as my mentor, Dr. Dixon Woodbury, pointed out the channels had been there before with those same SNAP-25 samples and we needed to see them and them reliably eliminate them to have a valuable control for my experiments.
Next, I ran an SDS-PAGE on each of my four samples to see if SNAP-25 oxidation could be detected. The samples were run in parallel lanes on the gel under alternating reduced and non-reduced conditions. The amount of oxidized SNAP-25 appeared to be similar under both reducing and non-reduced (presumably oxidized) conditions. It was also evident that SNAP-25 spontaneously oxidizes in solution given enough time since no oxidizing reagent was used to induce the formation of SNAP-25 aggregates.
Seeing that I couldn’t observe the masking SNAP-25 channels anymore, I decided to forge ahead and to attempt my original experiments. The first thing I had to do was to make artificial vesicles and to test for their fusigenic properties . Upon doing this, and in the absence of protein, I consistently saw the appearance of channels in the membranes. To eliminate the possibility of contamination from stock solutions of buffer, lipids and salt gradients, and contamination of the bilayer chambers and stir bars, I performed extensive controls adding in one variable at a time until I saw these odd channels. They only appeared when I added the artificial vesicles to the chamber. This suggested the existence of a confounding variable in the nystatin or ergosterol in the artificial vesicles. Since that is the basis of the method I intended to use to measure vesicle fusions my entire experimental protocol is invalidated. That concluded my work, just prior to my graduation from BYU, which cannot be continued until someone has isolated and verified the source of the contaminating channels in this system.
This taught me many valuable lessons about research. First, you can’t always predict exactly what is going to happen because you can’t always see all the variables. Second, isolating a causative variable with certainty requires having a very simple system over which you have ultimate control. Last, sometimes science is just hard, long, tedious work. I’ve begun to learn the importance of very meticulous note-taking and careful controls for all your experiments.
I’m very grateful for the research experience I had at BYU and for the opportunity to earn this ORCA grant. I’ve now graduated and am starting a doctoral program in pharmacology research in August 2004 at the University Of Texas Health Science Center at San Antonio. Receiving this grant has certainly opened the door for me to get into this research program. The mentoring program at BYU is exceptional. The opportunity to work closely with professors, like Dr. Woodbury, on cutting edge research projects was an invaluable part of my educational experience at BYU.
References
- Woodbury, D.J., K. Rognlien, 2000. The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes. Cell Biology International. 24:809-818.
- This data was presented in a poster at the 49th Annual Meeting of the Biophysical Society at Long Beach, CA in February 2005. I was one of the presenters of this poster titled “Dissecting the mechanism whereby native vesicles fuse to a syntaxin bilayer”. All the experiments leading to this poster were done in the lab, and under the direction, of Dr. Dixon Woodbury, Brigham Young University, Department of Physiology and Developmental Biology.
- Woodbury, D.J., 1999. Nystatin/Ergosterol Method for Reconstituting Ion Channels into Planar Lipid Bilayers. Methods in Enzymology. 294:319-339.