Using Spheroplasts to Study Peptide Interactions with Cell Membranes

Cytoplasmic membranes remain intact if bacteria cells are stripped off the outer cell wall and the peptidoglycan-they are called spheroplasts or protoplasts. This allows us to study the interaction of membrane-active antibiotics directly with the cytoplasmic membranes of bacterial. For our purpose, we want giant spheroplasts in order to apply the aspiration techniques. The technique of aspiration serves two purposes: one is to apply tension to the membrane and another is to measure the membrane area changes. The measurement of the membrane area change by tension or peptide binding or any structural event is the best physical quantitative description for the state of the membrane. We believe that these experiments will lead to discover the so-far unknown properties of cell membranes. Using established methods we grew spheroplasts which were stabilized in a STOP solution. To test the condition of the cytoplasmic membranes, we then diluted the STOP solution by adding pure water. We found that as the STOP solution was diluted, the water influx enlarged the spheroplasts. This seems to indicate that there is a membrane reservoir (perhaps, invaginations of cell membrane) in the original state of spheroplast. By the aspiration method, we measured the tension versus membrane area change of spheroplasts stabilized at 15% STOP solution. The tension vs. area change showed a slow exponential region followed by a linear region, similar to pure lipid GUVs. However the stretch expansion moduli are 5 times smaller than that of pure lipid bilayers. When maganin or melittin, were introduced into spheroplast suspension, we found that the surface area was expanded by more than 5% by peptide binding. These preliminary results indicate the feasibility of using spheroplasts as an experimental platform for studying the interaction of membrane-active peptides with cell membranes.