SPATIAL COMPARTMENTATION AND TIME RESOLUTION OF PHOTOOXIDATION OF A CELL-MEMBRANE PROBE IN ELECTROPERMEABILIZED CHINESE-HAMSTER OVARY CELLS

When electropulsed by short strong electric field pulses, cells can be permeabilized transiently (electropermeabilization). The transient electroinduced membrane restructuration which supports enhanced membrane permeability is topologically well defined on the surface of the cell. Exchange of polar species only takes place on the part of the cell surface which is controlled by the field intensity. Electropermeabilization is a stress for the cell and metabolic responses associated with this stress must be studied. A key point is to discover whether this stress affects the whole cell surface or especially the permeabilized part. This was investigated by photochemical time-dependent methodology. Analysis of the photooxidation reaction of 5-(N-hexadecanoyl)-aminofluorescein, an interfacial light-sensitive fluorescent probe, inserted into the Chinese hamster ovary cell membrane showed that the rate of reaction was accelerated when the cell population was electropermeabilized. This increase in the rate constant depended on the field strength. A theoretical approach suggested that modulation of the photooxidation reaction was restricted to the electropermeabilized part of the cell surface. The difference of reactivity could be correlated with an activator effect of oxygen-reactive species generated by an electroinduced oxidative jump and suggested that these species were localized in the permeabilized part of the plasma membrane. Using fluorescence digitized videomicroscopy imaging on a single cell, such spatial heterogeneity of the photooxidation reaction was indeed directly observed when the cell was electropermeabilized. There was heterogeneous compartmentation of the membrane reaction into two independent domains with specific reactivity. The direct observation is that long-lived electropermeabilization only affects a restricted part of the pulsed cell surface, from both a structural and a metabolic point of view.