Confocal microscopy investigations of phospholipase A2 induced deformation to giant lipid vesicles

Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of phospholipids present in the cellular membrane bilayer breaking them into fatty acids and lysolipids. These products perform physiological and patho-physiological functions. PLA2 activity has been the subject of research for a long time. However, PLA2 induced membrane deformation is an understudied effect. It is the focus of the present work. Effects of interaction between bee venom PLA2 and membrane-mimicking Giant Unilamellar Vesicles (GUV; diameter>5µm) of phosphatidylcholine (PC) lipids were investigated by Confocal Microscopy (CFM). GUVs were prepared by electroformation on indium tin oxide (ITO) slides. The bilayers were stained with a lipophilic dye to enable fluorescence imaging in CFM. CFM videos of the GUV/PLA2 reaction showed clear evidence of bilayer disruption due to hydrolysis. Inwardly directed tubules formed followed by GUV fragmentation at high PLA2 concentrations. Pores developed and passage of secondary structures into the vesicle was observed at medium concentrations. At lower concentrations GUVs shrank. In all cases, three stages could be identified: (i) apparent initial "inactive" period, (ii) damage development, and (ii) bursting. These results lead to the following hypotheses. At high and medium PLA2 concentrations hydrolysis product formation rate is faster than that of their lateral distribution, leading to product segregation. Furthermore, lysolipids remain in the outer monolayer, while some fatty acids translocate into the inner monolayer. Packaging of the dissimilarly shaped phospholipid (cylinder), lysolipid (cone) and fatty acid (inverted cone) in the asymmetric domains along with the requirement to shield the hydrophobic tails cause local stresses that drive invaginated tubules and pores. At lower enzyme concentrations, some bilayer relaxation occurs by formation of pores through which the enzyme enters into the GUV and hydrolyzes the inner monolayer leading to shrinkage. Such shrinkage has been reported for direct enzyme delivery inside the GUV but not for outside. Elucidating membrane damage is important because damages, such as pores, cause cell leakage, which can lead to cell death. Tubules and secondary structures are mechanisms of intercellular and intracellular transport. To the best of our knowledge, this is the first observation of such structures resulting from PLA2 activity.