BINDING OF FLUORESCENCE-LABELED AND GOLD-LABELED OLIGODEOXYRIBONUCLEOTIDES TO CYTOPLASMIC INTERMEDIATE FILAMENTS IN EPITHELIAL AND FIBROBLAST CELLS

Previously, in vitro experiments have demonstrated the capacity of int ermediate filaments (IFs) to associate with polyanionic compounds, inc luding nucleic acids. To prove that this activity is also shown by IFs in quasi-intact cells, digitonin-permeabilized epithelial PtK2 and mo use fibroblast cells were treated with FITC-labeled, single stranded o ligodeoxyribonucleotides and analyzed, after indirect decoration of th eir IF systems with TRITC-conjugated antibodies, by fluorescence micro scopy. While cytokeratin Ifs exhibited a strong affinity for and exact codistribution with oligo(dG)(25), vimentin IFs were less active in b inding this oligonucleotide. Other oligonucleotides, like oligo(dT)(25 ), oligo[d(GT)(12)G] and oligo[d(G(3)T(2)A)(4)G], were bound to IE's w ith lower efficiency. In general, the introduction of dA residues into oligo(dG), or oligo(dGT)(n) tracts reduced the IF-binding potential o f the nucleic acids. This, however, increased significantly upon reduc tion of the ionic strength to half physiological, indicating a strong electrostatic binding component. The binding reaction was often obscur ed by simultaneous association of the oligonucleotides with cellular m embranes mostly in the perinuclear region, an activity that was largel y abolished by prior cell extraction with nonionic detergent. Strongly IF-binding oligonucleotides also disassembled microtubules, presumabl y via their interaction with microtubule-associated proteins, but left microfilaments intact. In PtK2 cells, oligo(dG)(25)-loaded IFs were f requently seen coaligned with microfilaments and to cross-bridge stres s fibers with the formation of rope ladder-like configurations. Employ ing 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 ele ctron microscopy of permeabilized cells treated with gold-conjugated o ligonucleotides revealed decoration of Ifs and membrane systems with g old particles, whereby in PtK2 cells these structures showed a distinc tly heavier labeling than in fibroblasts. These results demonstrate th at in animal cells Ifs are able to bind nucleic acids and, very likely , also nucleoprotein particles and suggest that this capacity is explo ited by the cells for transient storage and, in cooperation with micro tubules and microfilaments, controlled transport of such material in t he cytoplasm. (C) 1997 Academic Press.