MICROINJECTION INTO GIANT VESICLES AND LIGHT-MICROSCOPY INVESTIGATIONOF ENZYME-MEDIATED VESICLE TRANSFORMATIONS

Background: 'Giant vesicles' have diameters of several micrometers and can be observed by Light microscopy. Their size may allow manipulatio n of individual vesicles and direct observation of the progress of a c hemical reaction in real time. We set out to test this possibility usi ng enzymatic hydrolysis of vesicle components as a model system. Resul ts: We describe a novel micromanipulation technique that allows us to microinject femtoliter amounts of a reagent solution adjacent to or in to giant vesicles with diameters ranging from 10 to 60 mu m. The vesic le transformations can be monitored directly in real time by light mic roscopy and recorded by video analysis. Snake venom phospholipase A(2) was added to vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3- phosphocholine, and the enzymatic hydrolysis of components of the lipi d bilayer was observed over time. A specific effect on the targeted gi ant vesicle was seen and video recorded, while the neighbouring vesicl es remained unaffected. Addition of the enzyme to the outside of a ves icle caused it to burst, whereas injection of the enzyme inside a vesi cle resulted in a slow and constant decrease in its size, until it eve ntually disappeared from the resolution power of the light microscope. Conclusions: These results show that it is possible to micromanipulat e an individual vesicle, and to follow visually the progress of an enz ymatic reaction occurring on the vesicle bilayer over time.