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...
Diethylene glycol distearate is used as a removable embedding medium to produce embeddment-free sections for transmission electron microscopy. The easily cut sections of this material float and form ribbons in a water-filled knife trough and exhibit interference colors that aid in the selection of sections of equal thickness . The images obtained with embeddment-free sections are compared with those from the more conventional epoxyembedded sections, and illustrate that embedding medium can obscure important biological structures, especially protein filament networks. The embeddment-free section methodology is well suited for morphological studies of cytoskeletal preparations obtained by extraction of cells with nonionic detergent in cytoskeletal stabilizing medium. The embeddment-free section also serves to bridge the very different images afforded by embedded sections and unembedded whole mounts .Transmission electron microscopic (TEM)" images of conventional thin sections are influenced by the presence of the embedding material to a degree not often appreciated . The embedding resin, especially epoxy, scatters electrons very much the way the embedded specimen does, rendering it nearly invisible, except where heavy-metal stains are bound (9) . The thin section is most effectively stained at its surface (I1, 18) and is thus essentially a two-dimensional slice of three-dimensional objects whose actual morphology can only be reconstructed with difficulty. Such images formed from exceedingly thin planes are excellent for visualizing many biological structures but are inappropriate for examining three-dimensional networks of protein filaments (5) such as those constituting the cytoskeletal framework (i.e., surface lamina and a protein filament network surrounding the chromatin-containing nucleus : reference 13) .An alternate but under utilized method of electron microscopy, the unembedded whole mount, provides an image 'Abbreviations used in this paper: DGD, diethylene glycol distearate ; MDCK, Madin-Darby canine kidney; nBA, n-butyl alcohol; TEM, transmission electron microscopy . directly from the specimen without resort to heavy-metal stains . In this procedure, the entire fixed, dehydrated, and critical-point dried cell is placed in the electron beam path in vacuo and biological material scatters electrons sufficiently to form very high contrast images. The important differences between stained, embedded sections and unstained whole mounts has been discussed in detail by Wolosewick and Porter (23,24) for unextracted, fixed (i.e., intact) cells .The embeddment-free whole mount has proven essential to the study of the cell architecture remaining after cells have been extracted with nonionic detergent. This procedure removes most lipids and soluble components leaving the cytoskeletal framework (for reviews see references 13 and 14) . This entity, revealed by detergent extraction, retains the configuration of the unextracted cell . Such preparations appear quite empty of structural elements when viewed in ...
Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections . The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100 . The cytoskeleton was further fractionated by extractionwith (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei . A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrixintermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions . Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique . Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct. Sequential Extraction of Cytoskeletal ElementsWhole mounts of detergent-extracted cells have proved to be well suited to the study of cytoskeletal organization (1-9). In the absence ofembedding plastic, the cytoskeletal filaments remaining after the removal of phospholipid and soluble proteins form clear images in transmission electron micrographs without the need for heavy metal staining. These images provide insights into the composition and organization of cytoplasmic filament networks that appear intimately associated with both the nucleus (9, 10) and the plasma lamina (11).In initial extraction, Triton X-100 in a buffer designed to best preserve the architectural elements of the cell is used. However, further extraction of the cytoskeleton reveals both biochemically and morphologically important substructures of the cytoskeleton and nuclear matrix . Most of the cytoskeleton is removed by extraction with (NH,)2S0,, leaving the network of intermediate filaments anchored to the nucleus. The nucleus itself is subfractionated by removal of the chromatin, a procedure that reveals the dense fibers of the nuclear lamina and the internal matrix (unpublished observations).The nuclear matrix-intermediate filament structure is resistant to high salt and is clearly defined...
Infection of HeLa cells with adenovirus serotype 2 causes rearrangements in nuclear matrix morphology which can best be seen by gentle cell extraction and embedment-free section electron microscopy. We used these techniques to examine the nuclear matrices and cytoskeletons of cells at 6, 13, 28, and 44 h after infection. As infection progressed, chromatin condensed onto the nucleoli and the nuclear lamina. Virus-related inclusions appeared in the nucleus, where they partitioned with the nuclear matrix. These virus centers consisted of at least three distinguishable areas: amorphously dense regions, granular regions whose granulations appeared to be viral capsids, and filaments connecting these regions to each other and to the nuclear lamina. The filaments became decorated with viral capsids of two different densities, which may be empty capsid shells and capsids with DNA-protein cores. The interaction of some capsids with the ifiaments persisted even after lysis of the cell. We propose that granulated virus-related structures are sites of capsid assembly and storage and that the filaments may be involved in the transport of capsids and capsid intermediates. The nuclear lamina became increasingly crenated after infection, with some extensions appearing to bud off and form blebs of nuclear material in the cytoplasm. The perinuclear cytoskeleton became rearranged after infection, forming a corona of decreased filament number around the nucleus. In summary, we propose that adenovirus rearranges the nuclear matrix and cytoskeleton to support its own replication.
The association of poliovirus metabolism with the cytoskeleton was investigated. Infected cells were extracted by using the nonionic detergent Triton X-100 in the physiological cytoskeleton buffer. The skeletal framework obtained was examined by transmission electron microscopy of resinless sections. The fibers of the framework were grossly distorted in infected cells. No virions or procapsids were seen but many virus-specific spheroidal bodies were associated with the framework. They had a diameter of 40 to 70 nm, were characterized by a dense core and a translucent periphery, and occurred in strings, often near the remnants of flattened vesicles. These spheres may correspond to virus-synthesizing bodies. The metabolism of poliovirus RNA was shown to be associated with the skeletal. framework by pulse-labeling cells with [3HJuridine and measuring the RNA retained on the framework. 20S double-stranded RNA, a form of poliovirus RNA found only in the replication complex, was attached to the skeleton throughout a 60-min pulse-label. 35S single-stranded viral RNA, a form found in virions, in polyribosomes, and in the replication complex, appeared first on the framework but after a few minutes was also found in the soluble cytoplasmic phase, encapsidated in virions. In contrast to viral RNA, viral proteins exhibited a varied association with the skeletal framework. Viral proteins were pulse-labeled with [35S]methionine and chased with unlabeled methionine. Although all of the virus-specific proteins were found, to some extent, in the skeletal fraction, the derivatives of P2 (P2-X and P2-5) and a derivative of P3 (P3.2) showed a preferential association with the skeletal framework. Virions and procapsids, on the other hand, were not associated with the cytoskeleton; both they and their component proteins (PlVPO, P1-VP1, P1-VP2, and P1-VP3) were found dominantly in the soluble cytoplasmic phase. The pathway of poliovirus assembly can be inferred from the above data. It is different from that found previously for the enveloped vesicular stomatitis virus and may be representative of encapsidated cytoplasmic virus assembly.
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