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Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Krauss SW, Larabell CA, Lockett S, Gascard P, Penman S, Mohandas N, Chasis JA - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, 94720, USA.

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.

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Immunolocalization of protein 4.1 in nuclear matrix visualized by high-resolution resinless electron microscopy. CaSki cells  were extracted with 0.5% Triton X-100 in CSK buffer, chromatin was removed by HaeIII and PstI digestion, and proteins were extracted with 0.25 M ammonium sulfate followed by extraction with 2 M NaCl. After fixation, the matrix preparation was incubated with  protein 4.1 antibody 24-3 (A) and 24-2 (B) and then with colloidal gold–coupled secondary antibody. The EM micrograph of a resinless  section shows a network of nuclear matrix core filaments (CF) and dense bodies (DB). The 10-nm gold beads decorated principally the  periphery and some internal areas of the dense bodies (arrowheads). This localization pattern at dense bodies in the matrix was also observed in a parallel experiment using anti–10-1 IgG but not with control IgG. Bar, 100 nm.
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Figure 6: Immunolocalization of protein 4.1 in nuclear matrix visualized by high-resolution resinless electron microscopy. CaSki cells were extracted with 0.5% Triton X-100 in CSK buffer, chromatin was removed by HaeIII and PstI digestion, and proteins were extracted with 0.25 M ammonium sulfate followed by extraction with 2 M NaCl. After fixation, the matrix preparation was incubated with protein 4.1 antibody 24-3 (A) and 24-2 (B) and then with colloidal gold–coupled secondary antibody. The EM micrograph of a resinless section shows a network of nuclear matrix core filaments (CF) and dense bodies (DB). The 10-nm gold beads decorated principally the periphery and some internal areas of the dense bodies (arrowheads). This localization pattern at dense bodies in the matrix was also observed in a parallel experiment using anti–10-1 IgG but not with control IgG. Bar, 100 nm.

Mentions: Electron microscopy was used to achieve greater resolution in localizing 4.1 epitopes in the nucleus. Cells growing on coverslips were prepared for embedment-free (“resinless”) section electron microscopy, a technique developed specifically to image three-dimensional cytoplasmic and nuclear structures normally concealed in conventionally embedded samples (Capco et al., 1984; Nickerson and Penman, 1992; Nickerson et al., 1994). Samples were sequentially extracted, lightly fixed, and incubated with primary IgGs against 4.1 peptides or normal rabbit IgG. A secondary antibody coupled to gold beads was added, and the preparation was fixed again and then embedded and sectioned. After removal of the temporary embedding material, epitopes for 4.1 were detected within the residual nuclear matrix structures visualized by electron microscopy (Fig. 6), consistent with the results obtained with immunofluorescence assays run in parallel samples. At the higher resolution of electron microscopy, the 4.1 epitopes retained in nuclear matrix preparations were associated with dense bodies and not with the supporting filamentous network. Parallel samples incubated with control IgG did not show labeling of nuclear structures.


Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Krauss SW, Larabell CA, Lockett S, Gascard P, Penman S, Mohandas N, Chasis JA - J. Cell Biol. (1997)

Immunolocalization of protein 4.1 in nuclear matrix visualized by high-resolution resinless electron microscopy. CaSki cells  were extracted with 0.5% Triton X-100 in CSK buffer, chromatin was removed by HaeIII and PstI digestion, and proteins were extracted with 0.25 M ammonium sulfate followed by extraction with 2 M NaCl. After fixation, the matrix preparation was incubated with  protein 4.1 antibody 24-3 (A) and 24-2 (B) and then with colloidal gold–coupled secondary antibody. The EM micrograph of a resinless  section shows a network of nuclear matrix core filaments (CF) and dense bodies (DB). The 10-nm gold beads decorated principally the  periphery and some internal areas of the dense bodies (arrowheads). This localization pattern at dense bodies in the matrix was also observed in a parallel experiment using anti–10-1 IgG but not with control IgG. Bar, 100 nm.
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Related In: Results  -  Collection

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Figure 6: Immunolocalization of protein 4.1 in nuclear matrix visualized by high-resolution resinless electron microscopy. CaSki cells were extracted with 0.5% Triton X-100 in CSK buffer, chromatin was removed by HaeIII and PstI digestion, and proteins were extracted with 0.25 M ammonium sulfate followed by extraction with 2 M NaCl. After fixation, the matrix preparation was incubated with protein 4.1 antibody 24-3 (A) and 24-2 (B) and then with colloidal gold–coupled secondary antibody. The EM micrograph of a resinless section shows a network of nuclear matrix core filaments (CF) and dense bodies (DB). The 10-nm gold beads decorated principally the periphery and some internal areas of the dense bodies (arrowheads). This localization pattern at dense bodies in the matrix was also observed in a parallel experiment using anti–10-1 IgG but not with control IgG. Bar, 100 nm.
Mentions: Electron microscopy was used to achieve greater resolution in localizing 4.1 epitopes in the nucleus. Cells growing on coverslips were prepared for embedment-free (“resinless”) section electron microscopy, a technique developed specifically to image three-dimensional cytoplasmic and nuclear structures normally concealed in conventionally embedded samples (Capco et al., 1984; Nickerson and Penman, 1992; Nickerson et al., 1994). Samples were sequentially extracted, lightly fixed, and incubated with primary IgGs against 4.1 peptides or normal rabbit IgG. A secondary antibody coupled to gold beads was added, and the preparation was fixed again and then embedded and sectioned. After removal of the temporary embedding material, epitopes for 4.1 were detected within the residual nuclear matrix structures visualized by electron microscopy (Fig. 6), consistent with the results obtained with immunofluorescence assays run in parallel samples. At the higher resolution of electron microscopy, the 4.1 epitopes retained in nuclear matrix preparations were associated with dense bodies and not with the supporting filamentous network. Parallel samples incubated with control IgG did not show labeling of nuclear structures.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, 94720, USA.

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.

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