Biomedical subjects

G Krochmalnic

Publications and source records attributed to G Krochmalnic.

7 recordsLinked to original sources

PubMed1989

Chromatin architecture and nuclear RNA.

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined 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 clumps, 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-beta-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.

Cell Nucleus↗

PubMed1987

Alterations in nuclear matrix structure after adenovirus infection.

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 filaments 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.

Adenoviruses, Human↗

PubMed1986

Resinless section electron microscopy of HeLa cell mitotic architecture.

The use of resinless sections extends embedment-free electron microscopy to the cytoskeleton of thick specimens. Here we examine HeLa cells rounded at mitosis. Extraction of mitotic HeLa cells with Triton X-100 removes lipids and soluble proteins, leaving the cytoskeletal framework and spindle apparatus. After fixation, the samples are embedded and sectioned, and the temporary embedding resin is removed for direct visualization in the electron microscope. The micrographs show that the cytoskeletal framework, chromosomes, spindle, and centrioles form an interconnected entity. The pericentriolar region, indistinct in conventional micrographs, appears composed of distinct fibers interconnecting the spindle microtubules and centriole. The resinless sections also reveal characteristic lacunae at late anaphase/early telophase. These probably result from reformation of the interphase cytoskeleton lagging reassembly of the nucleus.

Centrioles↗

PubMed1986

The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Fractionation↗

PubMed1985

Poliovirus metabolism and the cytoskeletal framework: detergent extraction and resinless section electron microscopy.

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 [3H]uridine 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 (P1-VP0, 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.

Capsid↗

PubMed1984

A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks.

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 epoxy-embedded 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.

Agar↗

PubMed1984

Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

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 extraction with (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 matrix intermediate 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.

Animals↗