Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization . We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis . First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer . The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely 'unaltered by detergent extraction . Residual desmosomes, as can be seen in thin sections, were also well-preserved . The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation . The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders . This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts . Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold . Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders . The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions . The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold .A potentially powerful addition to the study of cell structure is afforded by combining detergent extraction with unembedded whole mount electron microscopy. In this protocol, soluble proteins are extracted with non-ionic detergent under near physiological conditions of ionic strength and pH. The cellular structure that remains after detergent extraction is called the skeletal framework (1, 2). The elaborate filament framework can be clearly seen using whole mount transmission electron microscopy, which omits conventional embedding, sectioning, and staining. We have observed the three-

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The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.Nucleic acid metabolism is spatially organized in the cell nucleus. The application of powerful microscopy techniques has revealed an increasingly intricate domain organization within the nucleus (reviewed in ref. 1). Individual catalytic processes and the machinery they require are structurally constrained to spatial domains. The very intricate spatial organization of the nucleus presents an important research problem. Our goals are to identify the structure(s) maintaining the complex architecture of the nucleus and to characterize the molecular interactions that constrain components to specific locations.Much of the domain organization of the nucleus remains after the experimental removal of chromatin (2-9). This suggests that chromatin itself is not the fundamental structure organizing the nucleus. In fact, chromatin may itself be architecturally organized in loop domains attached at their bases to an underlying structure (10, 11).There is a second nucleic acid-containing structure distributed throughout the nucleus, an ribonucleoprotein (RNP)-containing network of fibrils and granules selectively stained by the EDTA-regressive method (12)(13)(14). This structure, identified in intact nuclei, corresponds to the nuclear matrix remaining after biochemical fractionation (15-17). The isolated nuclear matrix retains most nuclear RNA (15, 18), RNP proteins (17, 19), and may even require intact RNA for structural integrity (19,20). It is this nuclear matrix to which chromatin loops are anchored (11,21,22).Biochemical studies of the nuclear matrix and detailed ultrastructural studies of its architecture required the development of techniques to remove the larger mass chromatin while leaving the nuclear matrix undisturbed. Two very different and somewhat harsh fractionation protocols have uncovered a network of highly branched 10-nm filaments connected to the nuclear lamina and well distributed through the nuclear volume (18,23). These may form the core structure upon which the RNP-contain...

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have exmned the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large dumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-,-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.The eukaryotic nucleus remarkably packages more than a yard of DNA into a 5-,um spheroid. The packaging of DNA into chromatin and of chromatin into the nucleus is highly ordered (1-6), with the coarse features of this organization correlating with transcriptional activity. The condensed chromatin (heterochromatin) is largely inactive, and transcription is localized in the extended, dispersed euchromatin. The basic packing structures, the nucleosomes, are arranged in polynucleosome chains, which are wound into 30-nm fibers (7). Much less is known about the packing of chromatin fibers into higher order structures in the nuclear interior.There is a clear dependence of chromatin architecture on ongoing RNA metabolism; inhibition of RNA synthesis results in the retraction of chromatin from the nuclear lamina and its aggregation into massive clumps (8), whereas the spatial distribution of stained DNA in high-salt-extracted nuclei is changed by treatment with RNase (9). In what may be a related phenomenon, RNA synthesis inhibitors cause a retraction of polytene chromosome puffs (10,11).Many studies have described an internal structural framework in the nucleus called the nuclear matrix (12)(13)(14)(15)(16)(17), which is associated with many important chromatin functions, including DNA replication (18, 19), RNA synthesis and p...