Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function.
The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.
Tolstonog
Genrich V
GV
Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany.
Sabasch
Michael
M
Traub
Peter
P
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis*; D007382:Intermediate Filaments / Q000502:physiology / Q000648:ultrastructure*; D007633:Keratins / Q000032:analysis; D034921:Lamin Type B; D034882:Lamins; D051379:Mice; D008856:Microscopy, Fluorescence; D015530:Nuclear Matrix / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis / Q000648:ultrastructure; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000032:analysis / Q000648:ultrastructure
D007381:Intermediate Filament Proteins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D014746:Vimentin; D007633:Keratins; D004247:DNA