Abstract. The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged.Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E.G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins.Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles. This RNP-depleted nuclear matrix is markedly distorted in overall shape when compared to the RNP-containing nuclear matrix.C OMPARED to the detailed knowledge of the arrangements of defined sequences within DNA, little is known of its orga'nization in the nucleus. The interior of the eukaryotic nucleus and its composition has been particularly difficult to study. There is strong evidence of a nuclear skeleton or matrix, but its nature a...

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-

The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding runt domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/runt domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/runt domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a runt domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene.

The low abundance proteins of the nuclear matrix (NM) were separated from the intermediate filament (IF) (1-3, 8, 11-14, 17-19). The detection of the latter was somewhat dependent on the conditions of preparation as summarized by Berezney (20). The nuclear lamins, described by Gerace, Blobel, and others (21-25), are major components of the NM as prepared by different methods from a variety of cell types (10,(12)(13)(14)(17)(18)(19). There are nuclear components associated with but probably distinct from the NM. These include heterogeneous nuclear RNA (hnRNA) and the proteins of the RNP complex (26,27), whose extensive association with the NM (4, 14, 28-36) may reflect the proposed role of the NM in RNA splicing (37). Also, the intermediate filaments (IF) are tightly associated with the NM. Together, the matrix and IF constitute, in in situ preparations, a structural complex, which has been designated the NM-IF scaffold (11,14,19). In contrast to structures associated with the matrix, there is another class of fibers in the matrix interior composed of a discrete set of poorly characterized nonhistone nuclear proteins, which appear to be constituents of the matrix itself (3, 4, 8, 10-12, 14, 17, 38-40

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