Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes.
Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.
Zaitsev
Sergei
S
Department of Nephrology, Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Buchwalow
Igor
I
Haberland
Annekathrin
A
Tkachuk
Sergei
S
Zaitseva
Ioulia
I
Haller
Hermann
H
Böttger
Michael
M
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002401:Cathepsin B / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D004247:DNA; D002401:Cathepsin B; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin; D002118:Calcium