Erythrocyte-mediated microinjection of horseradish peroxidase into BHK cells

In order to establish the distribution with time of proteins microinjected into mammalian cells, horseradish peroxidase (HRP) was microinjected into baby hamster kidney (BHK) cells using chicken erythrocyte ghosts. At time intervals following initiation of fusion between ghosts and target cells, samples were fixed with aldehydes and the peroxidase visualized by reaction with diaminobenzidine and viewing by light and electron microscopy. At 10 min, the reaction product was observed within the cytoplasm of 60% of the microinjected cells, but was excluded from the nucleus and membranous organelles. In the other 40% of microinjected cells, the reaction product was also observed within the nucleus. At 30 min, the reaction product was observed to be evenly distributed throughout the cell, including the nucleus but excluded from organelles. By 6 h, the reaction product was present almost exclusively within the nucleus of 63% of microinjected cells. At all time points, 20–30% of the erythrocytes ghosts appear to have been taken up by cells by phagocytosis rather than fusion, as evidenced by the presence of peroxidase reaction product within intact and fragmented erythrocyte ghosts in the cytoplasm of target cells. Cells incubated with a lanthanum solution following fusion excluded this electron dense tracer, indicating that the cytoplasmic compartment is not opened during exposure to polyethylene glycol.