Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts

Abstract

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either (i) never greater than 0.5 nm in radius, (ii) much smaller in number if they were greater than 0.5 nm in radius, or (iii) shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.Electroporation Electropore Pore

Electroporation: High frequency of occurrence of a transient high‐permeability state in erythrocytes and intact yeast

“…RBCs are terminally differentiated, cannot be tested for growth, do not have DNA and RNA for PI staining, but are observed by us using microscopy to form spherical 'ghosts' [21] if there is significant FITC-dextran uptake. For the yeasts average viability was determined by direct plating.…”

“…Yeast were grown in YPD medium, harvested while in exponential phase and resuspended in a minimal medium (50 mM Na + 50 mM K phosphate buffer, pH 6.8, 1 mM glucose, 5 mM NH4C1) and 0.1 mM FITC-dextran (green fluorescence, GF; 0.01 tool FITC per tool glucose; 70 kDa, Sigma; effective radius about 5.8 nm) [21]. Control aliquots were removed before and after the electrical pulse experiments, corresponding to exposure of 5 and 40 min to the FITC-dextran.…”

“…However, previous ex- For example, successful introduction of DNA by electroporation not only requires uptake, but also non-degradative intracellular transport, correct incorporation and expression, and such experiments score only the small number of eventual transformants. Other studies are based on the average molecular uptake (or release) by an entire cell population [1,4,5] and give the average response of the cell population, while still other studies monitor the uptake or release of fluorescence by a single cell [20,21]. In contrast, we determine how many cells experience electroporation by measuring uptake of fluorescent test molecules by each cell within a large, statistically significant population.…”

“…[4,5,21,22], and intact yeast are of interest because they are readily tested for viability by plating, and because the creation and use of protoplasts [9,14] can be avoided [8,11] for introduction of macromolecules if electroporative uptake occurs with high frequency in intact cells. The morphology of RBCs and the two yeasts differs significantly: (i) an RBC is a 7/zm biconcave disc, (ii) Saccharomyces cerevisiae is an approx.…”

“…7 #m spheroid, and (iii) Schizosaccharomyces pombe is a rod about 3-5/zm in diameter and 5-15 pm in length [23]. In addition to studying cells with different properties, we used the test macromolecule, FITC-dextran, which has relatively small electrical charge, and which is therefore not strongly excluded from pores because of a Born energy interaction [21,24,25]. For this reason, FITC-dextran is a good probe for openings or pores in cell membranes.…”