Kristen H. Parker and Dr. John D. Bell, Physiology and Developmental Biology
Our research team in Dr. Bell’s laboratory has focused primarily on studying interactions between cell membranes and the enzyme secretory phospholipase A2 (sPLA2). This enzyme is able to distinguish between healthy cells and foreign or damaged cells, which it hydrolyzes. The purpose of my individual project was to use a specific fluorescent probe to study the mechanism for the selective susceptibility and degradation of cell membranes by sPLA2.
My hypothesis was that the lipids in the cell membrane become less tightly packed during cell death, making them susceptible to hydrolysis by sPLA2. To study this, I used a fluorescent probe called Merocyanine 540 (MC540) to observe the changes in lipid packing of the cell membrane. Similar experiments had already been completed with erythrocytes, however, it was important to determine if the membrane changes that occur in nucleated cells would be comparable.
MC540 binds to the outer leaflet of cell membranes and shows a shift in emission maximum and an increase in fluorescence intensity when lipids are less tightly packed (Jensen et al, 2005).
I conducted experiments in which I used a spectrofluorometer to gather spectral data of S49 lymphoma cells after addition of MC540 and then after addition of ionomycin, which simulates cellular death. Treatment of cells with ionomycin caused a significant increase in fluorescence intensity and a shift in emission maximum from about 583 nm to 586 nm (Fig. 1). These observed membrane changes induced by ionomycin show that a decrease in membrane lipid packing occurs at cell death. The data from my experiments helped us know more about changes that occur in nucleated cell membranes at cell death, and helped to further clarify the mechanism of selective susceptibility of hydrolysis by sPLA2. Greater understanding of this process is important as there is considerable evidence that sPLA2 plays a role in several conditions including sepsis, inflammatory diseases and ischemia.
The experimental data I collected was an important component of the paper submitted by Dr. Bell and our research team in December 2006 for publication in Biophysical Journal. My research experience gained in Dr. Bell’s laboratory was invaluable to my education at BYU. I learned a great deal and have recently graduated and obtained employment based upon this previous laboratory experience.