A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.

Applications of electroporation of adherent cells in situ, on a partly conductive slide.

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells by in situ electroporation are described. Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. The migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

Electroporation of peptides into adherent cells in situ.

To study the effect of protein interactions in vivo upon cellular functions,such complexes may be disrupted through the introduction of peptides corresponding to the proteins' points of contact. In this communication a simple, rapid and reproducible procedure for peptide introduction into adherent cells by electroporation is described. Cells are grown on electrically conductive, optically transparent indium-tin oxide at the time of pulse delivery. Several electrode and slide configurations, necessary to obtain non-electroporated cells adjacent to the electroporated ones as a control, are outlined. Careful control of electric field strength achieved the introduction of the peptide into essentially 100% of the cells while this treatment caused no detectable disruption of their division cycle.

A novel technique for the study of intercellular, junctional communication: electroporation of adherent cells on a partly conductive slide.

One of the effects of neoplastic transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). The investigation of junctional permeability is usually conducted through the microinjection of the fluorescent dye, Lucifer yellow, followed by observation of its migration into neighboring cells. This is a time-consuming approach, requiring expensive equipment. To overcome these problems, a novel technique was devised which takes advantage of the ability of short electric pulses to create transient "pores" on the cell membrane through which Lucifer yellow can enter, simultaneously and into large numbers of cells, with minimal disturbance to cellular metabolism. Cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse was applied in the presence of Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area was microscopically observed under fluorescence illumination. Using this technique, we investigated the relationship between expression of the middle tumor antigen of polyoma virus (mT) and GJIC in two representative cell systems with different responses to mT. The results show that low mT expression levels, although unable to transform rat F111 cells fully, are able to interrupt GJIC. Although parts of this mechanism might be mediated through protein kinase C (PKC), mT appears to have additional functions. PKC, however, had the opposite effect upon junctional permeability in a clone of mouse NIH-3T3 fibroblasts; intercellular communication in these cells appears to require PKC activity.

Electroporation of adherent cells in situ.

A simple, rapid, and reproducible procedure for the introduction of macromolecules into adherent mammalian cells by electroporation is described. Cells were growing on a glass surface coated with electrically conductive, optically transparent indium-tin oxide at the time of pulse delivery. Several factors affected the optimal voltage for permeation of a given line including the metabolic state of the cells and their degree of spreading onto the conductive growth surface. Careful control of the electric field strength resulted in almost 100% of the cells containing introduced antibodies without any detectable change in the length of their division cycle. Higher voltages were required for the stable expression of DNA than for the introduction of antibodies, resulting in a significant rate of cell death.