OBSERVATION OF EXTREMELY HETEROGENEOUS ELECTROPORATIVE MOLECULAR UPTAKE BY SACCHAROMYCES-CEREVISIAE WHICH CHANGES WITH ELECTRIC-FIELD PULSE AMPLITUDE

Molecular uptake of a charged fluorescent molecule (calcein; 623 Da, z = -4) was quantitatively determined at the single cell level using now cytometry. Dilutely suspended cells were exposed to one exponential pulse (tau(p) approximate to 300 mu s) for different field strength values. For an asymmetric cell such as the yeast Saccharomyces cerevisiae a significant variation in the number of molecules taken up by individual cells was expected for physical reasons. By carrying out several thousand individual cell measurements for each pulse condition, we found that the number of molecules per cell varies significantly within the cell population, and that this population distribution changes markedly as the field strength is varied. Surprisingly, in spite of significant changes in this distribution with field strength, the average uptake per cell reaches a non-equilibrium plateau for which the uptake per cell is much smaller than the product of the mean cell volume and the supplied extracellular concentration. These observations of different field-dependent cell population distributions of uptake support the hypotheses that (1) electroporation is a transmembrane voltage-responsive phenomenon, so that cells of different sizes, shapes and orientation, respond differently to even a spatially uniform applied field, (2) population average measurements of electroporation behavior can be incomplete and misleading, and (3) transport of small charged molecules is due to electrophoresis through the pores of a dynamically changing pore population.