Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.
The possibility of introducing definite amounts of nonpermeant molecules into electropermeabilized living cells has been approached by quantifying the amounts of Lucifer Yellow (LY; a 457-Da highly fluorescent molecule) and Phytolacca americana (Pokeweed) antiviral protein (PAP; a 30,000-Da ribosome-inactivating protein) retained by the cells after closure of the electric-field-induced transient structures of permeation. Without the electropermeabilization, these two molecules enter the cell only in very small amounts by fluid-phase pinocytosis. Under our experimental conditions, using the NIH 3T3 cells, the intracellular LY concentration can reach a value equivalent to the extracellular concentration and can be regulated by controlling the external concentration. We describe the use of LY in a rapid and efficient test for the determination of the best electrical-shock conditions of other cell lines. After electropermeabilization, PAP is 2 X 10(5) times more cytotoxic. Its toxicity can be detected at external concentrations (10(-11) M) corresponding to less than 10 internalized molecules per electropermeabilized cell. Therefore, after electropermeabilization, the nonpermeant molecules have a direct access to the cytosol and the biological effect of nonpermeant substances can be revealed.
Mir
L M
LM
Laboratoire d'Oncologie Moléculaire, Institut Gustave Roussy, Villejuif, France.
Banoun
H
H
Paoletti
C
C
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003600:Cytosol; D004560:Electricity; D006801:Humans; D066298:In Vitro Techniques; D007546:Isoquinolines; D008564:Membrane Potentials; D051379:Mice; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1
D007546:Isoquinolines; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C017475:lucifer yellow; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein