Figure 3.

Characteristics of P=S ODN shuttling. Microinjection of 30 µM P=S ODN 12182–Texas Red and 70 kDa dextran–Cascade Blue into one nucleus of binucleate Ref52 cells pretreated as indicated below. After injection cells were incubated for another 2 h before fixation. The arrows point to the non-injected nucleus in each case. (A) Microinjection into chilled cells and incubation on ice after injection. Shuttling was temperature-dependent and strongly inhibited at 4°C. (B) Injection into cells under conditions of ATP depletion showed that nucleocytoplasmic migration of P=S ODN was energy-dependent. (C and D) Cells were pre-injected with 1 mg/ml fluorescein-conjugated WGA into the cytoplasm 30 min prior to oligonucleotide injection. Inhibition of shuttling by WGA suggested a nuclear pore complex-mediated transport process. (E and F) The labeled P=S ODN was injected together with 2 mM unlabeled P=S ODN 8424 into one nucleus of a binucleate cell which had been pre-injected into the cytoplasm with WGA–fluorescein. Detection in the non-injected nucleus despite treatment with WGA indicated a passive diffusion-driven migration of 12182–Texas Red and thus saturation of the active transport process by the excess of unlabeled P=S ODN. The fluorescent images for the P=S ODNs (A–C and E) and WGA (D and F) are shown. Bar 10 µm.