Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.

Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells.

A major nucleolar protein with a molecular weight of 100 kD is directly implicated in the transcription of pre-ribosomal RNA (pre-rRNA) and appears to be cleaved into specific maturation products during pre-ribosome biogenesis. Polyclonal antibodies which recognize the 100 kD protein and its products were used to determine the correlation between rDNA transcription and these proteins. Actinomycin D (AMD) was used to block selectively rDNA transcription (AMD 0.1 microgram/ml). Immunoperoxidase and immunogold staining were carried out in untreated and treated cells. Digitalization allowed the quantification of label according to the nucleolar components and the cellular areas. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 kD protein than the granular RNP component but both nucleolar components were positively immunostained. The distribution of the 100 kD protein was rapidly modified by AMD: loss of label occurred first in the dense fibrillar zone of the nucleolus, demonstrating the correlation between rDNA transcription and the presence of this protein. However, one part of the protein remains in the segregated nucleolus after 1 h of AMD treatment, thus supporting the structural function of this protein.