In vivo observation of a nuclear channel-like system: evidence for a distinct interchromosomal domain compartment in interphase cells.
We have investigated the interchromosomal domain compartment in living cells by transfecting cDNA coding for Xenopus vimentin, engineered to contain a nuclear localization signal (NLS), coupled to the green fluorescent protein. In human vimentin-free SW13 cells, this chimeric protein was deposited in body-like "dots" both at 37 degrees C, the nonpermissive temperature for assembly of the amphibian vimentin, and 28 degrees C, the optimal temperature for Xenopus vimentin assembly, indicating that the chimeric protein was assembly incompetent. However, when transfected into a subclone stably expressing Xenopus NLS-vimentin (SW13-SC), the chimeric protein incorporated, as a fluorescent tracer, into the structures formed by NLS-vimentin and allowed us to visualize the outgrowth of the vimentin fibers after a temperature shift to 28 degrees C in living cells. In particular, we followed the time-dependent outgrowth of fibers from nuclear dots, first connecting two dots each and with time three and more, eventually generating a spatially restricted fiber system consisting of few loop-like arrays traversing the nucleus. Virtually identical results were obtained when the temperature was lowered only to 30 and 32 degrees C, respectively. An engineered human NLS-vimentin, without need for temperature shift, formed seemingly identical patterns of nuclear fibrils at 37 degrees C in three additionally transfected human cell lines: MCF-7, PLC, and HeLa. When the epithelial cytokeratin pair 8 and 18 was expressed in the nucleus via an engineered NLS in the cytokeratin 18 gene, more network-like, extended filament arrays were generated. Notably, in cotransfection experiments with Xenopus NLS-vimentin, we observed that the formation of these cytokeratin networks at 37 degrees C initiated from dots that nearly entirely colocalized with the aggregated amphibian NLS-vimentin. After a shift to 28 degrees C, extending Xenopus NLS-vimentin and cytokeratin filaments frequently followed the same path through the nucleus. These data indicate that interphase cells contain a seemingly equivalent, accessible interchromosomal space.
Reichenzeller
M
M
Division of Cell Biology, Biomedical Structure Analysis Group, Division of Molecular Organization of Complex Genomes, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, D-69120, Germany.
Burzlaff
A
A
Lichter
P
P
Herrmann
H
H
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000306:Adrenal Cortex Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure; D002875:Chromosomes / Q000502:physiology* / Q000648:ultrastructure; D002877:Chromosomes, Human / Q000502:physiology / Q000648:ultrastructure; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D007633:Keratins / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000235:genetics / Q000502:physiology*; D014981:Xenopus
D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D049452:Green Fluorescent Proteins; D007633:Keratins