Binding of fluorescence- and gold-labeled oligodeoxyribonucleotides to cytoplasmic intermediate filaments in epithelial and fibroblast cells.
Previously, in vitro experiments have demonstrated the capacity of intermediate filaments (IFs) to associate with polyanionic compounds, including nucleic acids. To prove that this activity is also shown by IFs in quasi-intact cells, digitonin-permeabilized epithelial PtK2 and mouse fibroblast cells were treated with FITC-labeled, single-stranded oligodeoxyribonucleotides and analyzed, after indirect decoration of their IF systems with TRITC-conjugated antibodies, by fluorescence microscopy. While cytokeratin IFs exhibited a strong affinity for and exact codistribution with oligo(dG)25, vimentin IFs were less active in binding this oligonucleotide. Other oligonucleotides, like oligo(dT)25, oligo[d(GT)12G] and oligo[d(G3T2A)4G], were bound to IFs with lower efficiency. In general, the introduction of dA residues into oligo(dG)n or oligo(dGT)n tracts reduced the IF-binding potential of the nucleic acids. This, however, increased significantly upon reduction of the ionic strength to half physiological, indicating a strong electrostatic binding component. The binding reaction was often obscured by simultaneous association of the oligonucleotides with cellular membranes mostly in the perinuclear region, an activity that was largely abolished by prior cell extraction with nonionic detergent. Strongly IF-binding oligonucleotides also disassembled microtubules, presumably via their interaction with microtubule-associated proteins, but left microfilaments intact. In PtK2 cells, oligo(dG)25-loaded IFs were frequently seen coaligned with microfilaments and to cross-bridge stress fibers with the formation of rope ladder-like configurations. Employing microinjection and confocal laser scanning microscopy, association of IFs with oligonucleotides could also be visualized in intact cells. In accord with these fluorescence microscopic data, transmission electron microscopy of permeabilized cells treated with gold-conjugated oligonucleotides revealed decoration of IFs and membrane systems with gold particles, whereby in PtK2 cells these structures showed a distinctly heavier labeling than in fibroblasts. These results demonstrate that in animal cells IFs are able to bind nucleic acids and, very likely, also nucleoprotein particles and suggest that this capacity is exploited by the cells for transient storage and, in cooperation with microtubules and microfilaments, controlled transport of such material in the cytoplasm.
Hartig
R
R
Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg/Heidelberg, Germany.
Huang
Y
Y
Janetzko
A
A
Shoeman
R
R
Grüb
S
S
Traub
P
P
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D004848:Epithelium / Q000648:ultrastructure; D005347:Fibroblasts / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D017979:Gold Colloid; D006651:Histocytochemistry; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007614:Macropodidae; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009994:Osmolar Concentration; D055672:Static Electricity
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D017979:Gold Colloid; D009838:Oligodeoxyribonucleotides; D004247:DNA; D016650:Fluorescein-5-isothiocyanate