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

Molecular uptake of a charged fluorescent molecule (calcein; 623 Da, z = -4) was quantitatively determined at the single cell level using no w cytometry. Dilutely suspended cells were exposed to one exponential pulse (tau(p) approximate to 300 mu s) for different field strength va lues. For an asymmetric cell such as the yeast Saccharomyces cerevisia e a significant variation in the number of molecules taken up by indiv idual cells was expected for physical reasons. By carrying out several thousand individual cell measurements for each pulse condition, we fo und that the number of molecules per cell varies significantly within the cell population, and that this population distribution changes mar kedly as the field strength is varied. Surprisingly, in spite of signi ficant changes in this distribution with field strength, the average u ptake 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 differ ent field-dependent cell population distributions of uptake support th e hypotheses that (1) electroporation is a transmembrane voltage-respo nsive phenomenon, so that cells of different sizes, shapes and orienta tion, 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 molecule s is due to electrophoresis through the pores of a dynamically changin g pore population.