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 B1C8 monoclonal antibody detects a 180-kDa nuclear matrix-specific protein. The protein is a component of the dense, metabolically active bodies or assemblies revealed by resinless section electron microscopy of the nuclear matrix. These assemblies are scattered through the nuclear interior, enmeshed in a complex network of 11-nm filaments. Resinless section electron microscopy of immunogold-stained nuclear matrix preparations shows B1C8 located in many but apparently not all the assemblies. In this regard, the B1C8 antigen resembles previously studied nuclear matrix proteins such as the H1B2 protein. The speckled pattern of nuclear immunofluorescence by B1C8 reflects this labeling of the dense assemblies in the nuclear matrix. Somewhat unusual is the faint staining of cytoplasmic microtubules by B1C8, which appears to be due to a weakly cross-reacting protein. During cell division, the B1C8 antigen redistributed drastically, showing the dispersion of nuclear matrix assemblies at mitosis. Speckles of B1C8 fluorescence first coalesced at prophase within the nuclear interior and then scattered into numerous cytoplasmic speckles by prometaphase. At metaphase, the B1C8 speckled cytoplasmic staining had become even more widely distributed and finely grained. Also, intense labeling appeared at the mitotic pole and on the spindle fibers themselves. The reassembly of BiC8 antigens into larger cytoplasmic speckles began at anaphase and finally, at telophase, most B1C8 labeling redistributed into speckles in the re-forming nuclei.There is increasing evidence that most, and probably all, nuclear macromolecular metabolism is closely associated with the nuclear matrix. More recent evidence indicates that nuclear events are highly localized into metabolically active centers. DNA synthesis takes place in replication sites (1-3), heterogeneous nuclear RNA (hnRNA) processing, and RNA transport in spliceosomes that process hnRNA (4-6), while nucleoli have long been known as dense bodies that synthesize and process rRNA (7).We have previously shown that the nuclear matrix is composed of a profuse network of "core filaments" that enmesh many dense bodies ofdiverse sizes and morphologies (8-10). Conventional electron micrographs of the nucleus show mostly the dense chromatin. Removal of the chromatin reveals an extensive nonchromatin nuclear structure consisting of filaments and dense bodies. While visible in conventional micrographs, only resinless section electron microscopy shows the filaments and dense bodies with great clarity in three dimensions. Immunolocalization with monoclonal antibodies shows the dense bodies to be of complex protein composition and to be the principal sites of nuclear metabolism. Because of their various types, complex compositionThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.and variety of functions, we suggest that these dense b...

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thinsection electron-microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy.In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying.In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.The localization of specific proteins with labeled antibodies is a fundamental technique for exploring cell structure. Cytoskeletal filaments were first displayed by immunofluorescent staining (1-4), profoundly changing our understanding of cell structure. Antibody-based localization achieves its highest resolution with the electron microscope. In the conventional technique samples are embedded in a resin that is cut into ultrathin sections. Antibodies stain the section surface wherever antigenic epitopes are exposed. An important advance in the conventional technology was the use of colloidal gold-conjugated second antibodies (5-7). This methodology is well suited for viewing cross sections of cell organelles and membrane systems. It is, however, severely limited for structures such as the cytoskeleton or nuclear matrix, whose form is apparent only when seen in three dimensions.The conventional thin section is inadequate for imaging the structural networks of the cell, showing only that portion of the sample appearing at the section surface. This is because the embedding plastic presents the same average electron scattering cross section as biological materials and thus masks cellular components contained in the section interior. Heavy metals stain the section surface forming the actual image. Filaments of the structural networks are seen only in cross section unless, as rarely happens, they are tangent to the section surface. However, there is a simple and effective alternative to the embedded section. Cell structures form high-contrast, three-dimensio...

Abstract. mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed HlB2 antibody detected a protein of 240 kD in the nuclear matrix fractions . The HIB2 antigen was completely masked in immunofluorescently stained interphase cells . However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope . The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments . Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves .The HIB2 antigen showed remarkable behavior at mitosis . As cells approached prophase the antigen be-W HILE the importance of the nonchromatin matrix in nuclear function is increasingly recognized, characterization of the matrix has lagged behind awareness of its functions . Little is known about nuclear matrix proteins and essentially nothing about their assembly into nuclear structure. We have begun addressing these questions systematically using mAbs to individual nuclear matrix proteins . With these, we can determine the biochemical properties of individual proteins, their peptide sequence, and their localization in the matrix . These antibodies detect the rearrangements of nuclear matrix proteins during mitosis, yielding important information about the process ofmatrix disassembly and assembly.Knowledge of the nuclear matrix proteins is essential for understanding important aspects of nuclear metabolism . Specific nuclear matrix proteins must conduct the important activities of this structure . These include serving as the site of DNA replication (Berezney and Coffey, 1975 ;McCready et al., 1980;Pardoll et al., 1980), hnRNA processing (Zeitlin et al., 1987(Zeitlin et al., , 1989, and steroid hormone action (Simmen