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 cell s by transfecting cDNA coding for Xenopus vimentin, engineered to contain a nuclear localization signal (NLS), coupled to the green fluorescent protei n. In human vimentin-free SW13 cells, this chimeric protein was deposited i n body-like "dots" both at 37 degrees C, the nonpermissive temperature for assembly of the amphibian vimentin, and 28 degrees C, the optimal temperatu re for Xenopus vimentin assembly, indicating that the chimeric protein was assembly incompetent. However, when transfected into a subclone stably expr essing Xenopus NLS-vimentin (SW13-SC), the chimeric protein incorporated, a s a fluorescent tracer, into the structures formed by NLS-vimentin and allo wed us to visualize the outgrowth of the vimentin fibers after a temperatur e shift to 28 degrees C in living cells. In particular, we followed the tim e-dependent outgrowth of fibers from nuclear dots, first connecting two dot s each and with time three and more, eventually generating a spatially rest ricted fiber system consisting of few loop-like arrays traversing the nucle us. Virtually identical results were obtained when the temperature was lowe red only to 30 and 32 degrees C, respectively. An engineered human NLS-vime ntin, without need for temperature shift, formed seemingly identical patter ns of nuclear fibrils at 37 degrees C in three additionally transfected hum an 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 cytokerat in 18 gene, more network-like, extended filament arrays were generated. Not ably, 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 NL S-vimentin. After a shift to 28 degrees C, extending Xenopus NLS-vimentin a nd cytokeratin filaments frequently followed the same path through the nucl eus. These data indicate that interphase cells contain a seemingly equivale nt, accessible interchromosomal space. (C) 2000 Academic Press.