Brent Brimhall and Joseph Callister with Dr. John Bell, Zoology

Microvesicles are small membrane-bound spheres released from certain cells undergoing the decay process. Little is known today about their properties and function at the cellular level. It is hypothesized that microvesicles play a role in cancer metastasis through the action of metalloproteinases enclosed within these spheres. Metalloproteinases are enzymes that act to break up the extracellular matrix of cells. This matrix serves as a type of glue that holds groups of cells together. Without this extracellular matrix in place cells are allowed to break free and migrate to other places of the body. In the case of cancerous cells this process is called metastasis. It is thought that these metallo-proteinases can be released from microvesicles when their membranes are broken apart by another enzyme called phospholipase A2. At this point it is not known, however, by what means microvesicle membranes are susceptible to this enzyme. The intent of this present research is to study some basic properties of microvesicles in order to better understand the mechanism by which phospholipase A2 breaks apart their phospholipid bilayer membrane. The following properties will be examined: membrane fluidity, protein content, membrane orientation, and lipid content.

To determine the relative fluidity of microvesicle membranes we used two fluorescent probes called laurdan and bis-pyrene in conjunction with a machine called a spectrofluorometer. When added to a solution of pre-isolated microvesicles, laurdan and bis-pyrene bind in the middle of the phospholipid bilayer. When these solutions were placed in the spectrofluorometer we could determine relative membrane fluidity by measuring the light emission from these fluorescent probes. Further analyses were then performed to determine the relationship between microvesicle membrane fluidity and their susceptibility to PLA2.

In another phase of this research we set out to determine the relative protein content of microvesicle membranes and how that related to PLA2 susceptibility. To determine this we compared the light absorbency spectrum of diluted samples of microvesicles to a known concentration of bovine serum albumin. We used a machine called a spectrophotometer to measure the amount of light at 595 nanometers absorbed by these samples. By comparing the absorbencies of our microvesicle solutions to the standard protein we were able to calculate the relative protein content of our samples. We then compared protein content to susceptibility of PLA2.

The third part of our experiment dealt with determining the orientation of a phospholipid called phosphotidylserine in the microvesicle membrane. In normal cells of the human body phosphotidylserine is oriented on the inside layer of a cell’s membrane. The microvesicles we studied are released from human red blood cells, and we wanted to see whether the microvesicle membranes maintained the same orientation as red blood cell membranes with phosphotidylserine located towards the inside. Using a chemical called prothrombin and the help again of a spectrophotometer we were able to determine relative amount of phosphotidylserine exposure. Prothrombin binds to phosphotidylserine only when it is exposed to the outside of a cell membrane. We compared normal microvesicles to ones that had been lysed in liquid nitrogen (thereby allowing all phosphotidylserine to be exposed to prothrombin). We then tried to see whether the amount of phosphotidylserine exposed in microvesicles had any correlation to their PLA2 susceptibility.

Finally, we determined the relative amount of lipid content in samples of microvesicles using a process called thin layer chromatography. Again we compared these results to PLA2 susceptibility.

Unfortunately, the results obtained from these experiments weren’t as conclusive as we would have hoped. When we compared the four microvesicle properties examined above to how susceptible they were to the enzyme PLA2 we saw little correlation. For example, a higher protein content in the membrane did not show a higher PLA2 susceptibility, or vice versa. The property that correlated most closely dealt with membrane orientation. We found that higher phosphotidylserine exposure to the outside of microvesicle membranes seemed to indicate that they were more susceptible to PLA2. Even though statistical analysis of this correlation proved not very accurate, it correlated more closely than any of the other properties.

Overall, this project was very enjoyable for us and we learned much in the process. Further experimentation is being carried on in Dr. Bell’s lab to learn more about microvesicles. We hope that such studies will be valuable in future cancer research.