Quinn Bahm and Dr. John Bell, Physiology and Developmental Biology
The phospholipids that make up the cell membrane change in order and liquidity with temperature and sterol concentration. This change in phase can be recognized by using a fluorescent probe called nystatin. Nystatin is currently used pharmaceutically as a fungicide; it forms channels in membranes with ergosterol allowing water and ions to rush inside and rupture the fungal cell. Nystatin is assumed to bind to the membrane differently in the presence of cholesterol, as is the case with humans than in the presence of ergosterol, thus allowing the drug to destroy fungal cells while accumulating harmlessly on the surface of the human cells. By studying the differences between the two cases, a nystatin binding standard can be established. The purpose of this ORCA project was to shed light on the way nystatin reacts to artificial membranes with ergosterol incorporated among the phospholipids. By establishing a known standard for nystatin binding we can then use it to study membrane order change in the different life stages of a cell such as apoptosis.
In general, sterols act as a type of membrane glue to keep phospholipids more ordered and that order affects nystatin intensity. Nystatin is thought to be non-florescent in solution, therefore as it loses affinity to the membrane at high-temperature phases intensity decreases. We saw ergosterol percentage affect the phase change in membrane phospholipids. At higher concentrations of the sterol, there was less of an intensity drop at the phase change. This agrees with what we assume sterols do; they hold the membrane together. The intensity drop would be the greatest in the membrane that had the greatest change; whereas the membrane with the most stabilizing sterol would have the lowest amount of phospholipid order change and therefore have the lowest intensity drop. Perhaps a more interesting piece of data is the effect of ergosterol on the temperature at which the phase change occurred. Since ergosterol is a membrane stabilizer it would be assumed that the more ergosterol a membrane had, the higher temperature it would take to change the phase of the membrane phospholipids. The data did not agree with this assumption, in fact it suggested just the opposite. As ergosterol percentage increased, the phase change of the membrane phospholipids started at a lower temperature and covered a wider spread of degrees Celsius.
Our comparative analysis of the ergosterol data with that of a similar cholesterol experiment done in our lab, gave us a good look at the difference between the two sterols. Nystatin binding to the membrane appears to fluoresce in the same manner be it by aggregating on the surface or by forming channels. The difference fluorescently could be negligible; more of the same studies would be needed to give more statistically sound evidence. The relative similarity in normalized intensity could mean that nystatin molecules bind with one another in a set fashion, such as always arranging in the shape of a pore, but then attach to the respective sterols in very different ways.
Any differences in intensities between the two sterols could be due to pipetting. We used a very small amount of nystatin (4 μl) and ergosterol (2-16 μl). If we were to get 1 μl extra of ergosterol in our vesicle solution that would increase the final concentration of sterol by almost 2.5%. If we were to add too much nystatin the effects would be less extreme but still cause variance in fluorescent intensity. The 15% and 20% concentrations appear to differ in magnitude of intensity jump from before the phase change to after. We would need to run another experiment with new vesicles of both types to decipher any distinctions due to pipetting or other variables.
Data for our 5% ergosterol vesicles looked very different than that of our cholesterol data. Examination of original emission spectra appears very noisy and the intensities are very low. This suggests that during the creation of the vesicles our small amount of nystatin never made it into solution. A slight of hand could of left the miniscule droplet adhered high on the side of the test tube far from the level of the other liquids in the tube. New vesicles of the same concentration should be constructed and the experiment run again to give us the missing
data.
My experience during this project was very unique. I had to calculate concentration sheets from which I then put together the vesicles I used in this experiment. I had to problem solve when the computers were not working, I hired an assistant to aid in the entire process, I had to use past experiments to finalize my data in the same manner and then compare the results. Dr. Bell helped me stay independent on this project and because of that I learned more about what I was doing. Dr. Bell helped me when I really needed it but for the most part I had to figure things out on my own. When I first started this project Dr. Bell said something to me that defines this project’s experience, he said something to the effect of you usually learn the most when you have to struggle to figure stuff out for yourself. I did struggle and I learned much in doing so.
This experiment is just one picture in a photo mosaic of the characteristics of the drug Nystatin. The graph I was able to put together will be used to explain a bigger picture of Nystatin and to guide further research on this probe. A future paper that includes this experiments data will be submitted for publication to a professional journal such as Biochemistry or the Biophysical Journal.