[The characteristics of the transport into the nucleus of conjugates with normal and mutant nucleophilic sequences]. / Kharakteristika transporta v iadro kon''iugatov s normal'noi i mutantnoi nukleofil'nymi posledovatel'nostiami.

A study was made of the transport of conjugates of a fluorescently labeled protein and a synthetic peptide, corresponding to the nuclear localization sequence of the SV 40 large T-antigen, into the nuclei of cultured human (HeLa and A431) and murine (HER14) cells. A possibility for such conjugates to be transported into the nuclei of digitonin-permeabilized cells, without addition of exogenous cytosol, was demonstrated. A quantitative comparison of the transport levels of constructions with normal or altered (K128T) peptide sequence was performed, and a low selectivity of nuclear transport with this alteration in the digitonin-permeabilized cell system was revealed, whereas constructions with the mutant sequence, when injected into live cells, remained in the cytoplasm. ATP-dependent transport of constructions with the mutant sequence into permeabilized cell nuclei was demonstrated, with a considerable part of this transport being NEM-insensitive. A suggestion is put forward that there are several variants of the nucleophilic sequence containing protein transport in the digitonin-permeabilized cell system.

Electrical properties of cell pellets and cell electrofusion in a centrifuge.

A new approach is proposed for studying cell deformability by centrifugal force, electrical properties of cell membranes in a high electric field, and for performing efficient cell electrofusion. Suspensions of cells (L929 and four other cell types examined) are centrifuged in special chambers, thus forming compact cell pellets in the gap between the electrodes. The setup allows measurement of the pellet resistance and also the high-voltage pulse application during centrifugation. The pellet resistance increases sharply with the centripetal acceleration, which correlates with reduction of the cell pellet porosity due to cell compression and deformation. Experiments with cells pretreated with cytochalasin B or colcemid showed that cell deformability depends significantly on the state of cytoskeleton. When the voltage applied to the cell pellet exceeds a 'critical' value, electrical breakdown (poration) of cell membranes occurs. This is seen as a deflection in the I(V) curve for the cell pellet. The electropores formed during the breakdown reseal in several stages: the fastest takes 0.5-1 ms while the whole process completes in minutes. A novel effect of colloid-osmotic compression of cell pellets after electric cell permeabilization is described. Supercritical pulse application to the cell pellet during intensive centrifugation leads to massive cell fusion. The fusion index grows with the increase of centripetal acceleration, and drops drastically when the pulse is applied after the centrifuge is stopped. The colloid-osmotic pellet compression enhances the fusion efficiency. No fusion occurs when cells are brought in contact after the pulse treatment. The data suggest that tight intermembrane contact formed prior to pulse application is a prerequisite condition for efficient cell electrofusion. The capacities of the technique proposed and the mechanism of membrane electrofusion are discussed.

Electrofusion of fibroblasts on the porous membrane.

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.

Differential regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells with various proliferative potentials.

The regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells of various proliferative potential was studied. The following regularities were found: 1) Both immortalized and non-immortalized cells can efficiently reactivate DNA synthesis in erythrocyte nuclei. 2) Erythrocytes drastically inhibit the entry of non-malignant culture cell nuclei into the S-period, not acting upon DNA synthesis. 3) The inhibitory action is characteristic of erythrocytes from different stages of chicken ontogenesis (from 5-day-old embryos to the adult bird). 4) Malignant cells are completely refractory to the inhibitory action of erythrocytes. The ability of erythrocytes to inhibit the onset of replication in heterokaryons may be connected with the mechanisms of maintaining these terminally differentiated cells in a non-proliferating state.