Epithelial structure revealed by chemical dissection and unembedded electron microscopy

The Journal of Cell Biology (Impact Factor: 9.69). 08/1984; 99(1 Pt 2):203s-208s.
Source: PubMed

ABSTRACT 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.

1 Follower
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Almost three decades ago, we presented a model where the extracellular matrix (ECM) was postulated to influence gene expression and tissue-specificity through the action of ECM receptors and the cytoskeleton. This hypothesis implied that ECM molecules could signal to the nucleus and that the unit of function in higher organisms was not the cell alone, but the cell plus its microenvironment. We now know that ECM invokes changes in tissue and organ architecture and that tissue, cell, nuclear, and chromatin structure are changed profoundly as a result of and during malignant progression. Whereas some evidence has been generated for a link between ECM-induced alterations in tissue architecture and changes in both nuclear and chromatin organization, the manner by which these changes actively induce or repress gene expression in normal and malignant cells is a topic in need of further attention. Here, we will discuss some key findings that may provide insights into mechanisms through which ECM could influence gene transcription and how tumor cells acquire the ability to overcome these levels of control.
    Advances in Cancer Research 02/2007; 97:275-94. DOI:10.1016/S0065-230X(06)97012-2 · 4.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.
    The Journal of Cell Biology 05/1997; 137(2):275-89. · 9.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Structural protein 4.1, first identified as a

Preview (3 Sources)

Available from