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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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Dynamic redistribution of protein 4.1 antigens during  the cell cycle. CaSki cells were permeabilized with 0.5% Triton  X-100 in CSK buffer to remove membranes and soluble proteins  before formaldehyde fixation, incubated with DNase I, and extracted with 0.25 M ammonium sulfate. The cell preparations  were immunostained with protein 4.1 antibody 24-2 (A, C, E, and  G) and with B4A11 (B, D, F, and H), a monoclonal antibody  against a nuclear matrix protein that displays an intense nuclear  speckle pattern at interphase but is not detectable at mitosis (Blencowe et al., 1994). Micrograph pairs show the fluorescent pattern  with anti–24-2 (left) and B4A11 (right) of the same fields. Cell cycle stages were also confirmed by viewing cells using phase contrast microscopy (not shown). (A) In the center, a mitotic cell  (M) showed staining of the mitotic spindle with particularly  strong staining of the spindle poles. The mitotic cell is surrounded  by interphase cells. Note that at opposite sides of an interphase  nucleus (I), immunostained centrosomes (small spots) are visible.  The inset shows the mitotic spindle of another cell intensely immunolabeled by anti–10-1. (B–F) Epitopes for B4A11 have disappeared in mitotic cells, but a strong speckled staining pattern is  present in interphase nuclei. (C) In the center, a cell in anaphase  retained a high degree of 4.1 staining in the area of the condensed  chromosomes. (E) As the cells approached telophase and cytokinesis, the bridge between the intensely stained perichromosomal  regions became visible by antibody deposition. As the daughter  cells separated further apart (G), bright 4.1 staining appeared at  the midbody (arrow). In the companion B4A11 fields, diffuse  staining began to condense into a more focal pattern, foreshadowing the appearance of the prominent B4A11 speckles characteristic of interphase cells. Bar, 5 μm.
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Figure 9: Dynamic redistribution of protein 4.1 antigens during the cell cycle. CaSki cells were permeabilized with 0.5% Triton X-100 in CSK buffer to remove membranes and soluble proteins before formaldehyde fixation, incubated with DNase I, and extracted with 0.25 M ammonium sulfate. The cell preparations were immunostained with protein 4.1 antibody 24-2 (A, C, E, and G) and with B4A11 (B, D, F, and H), a monoclonal antibody against a nuclear matrix protein that displays an intense nuclear speckle pattern at interphase but is not detectable at mitosis (Blencowe et al., 1994). Micrograph pairs show the fluorescent pattern with anti–24-2 (left) and B4A11 (right) of the same fields. Cell cycle stages were also confirmed by viewing cells using phase contrast microscopy (not shown). (A) In the center, a mitotic cell (M) showed staining of the mitotic spindle with particularly strong staining of the spindle poles. The mitotic cell is surrounded by interphase cells. Note that at opposite sides of an interphase nucleus (I), immunostained centrosomes (small spots) are visible. The inset shows the mitotic spindle of another cell intensely immunolabeled by anti–10-1. (B–F) Epitopes for B4A11 have disappeared in mitotic cells, but a strong speckled staining pattern is present in interphase nuclei. (C) In the center, a cell in anaphase retained a high degree of 4.1 staining in the area of the condensed chromosomes. (E) As the cells approached telophase and cytokinesis, the bridge between the intensely stained perichromosomal regions became visible by antibody deposition. As the daughter cells separated further apart (G), bright 4.1 staining appeared at the midbody (arrow). In the companion B4A11 fields, diffuse staining began to condense into a more focal pattern, foreshadowing the appearance of the prominent B4A11 speckles characteristic of interphase cells. Bar, 5 μm.

Mentions: Using three different classical fixation techniques (paraformaldehyde, methanol, or acetone), prominent punctate staining in the nuclear area of WI38 fibroblasts was consistently observed by indirect immunofluorescence with each antibody. Some antibodies also generated a more diffuse nuclear staining pattern and several of the antibodies produced cytoplasmic staining (Fig. 3). Parallel samples incubated with control IgG did not produce nuclear immunofluorescence with any of these fixation methods (Fig. 3). Fibroblasts were also probed with preparations of 24-2 or 24-3 IgGs directly labeled with either of two different fluorophores; these cells displayed immunofluorescent patterns similar to those obtained by indirect methods (data not shown). Nuclear immunofluorescent staining was also observed when another human fibroblast line (HCA), a transformed epithelial human line (CaSki; see Fig. 9) and a transformed murine fibroblast line (3T3; see Fig. 8) were probed with the 4.1 antibody panel.


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)

Dynamic redistribution of protein 4.1 antigens during  the cell cycle. CaSki cells were permeabilized with 0.5% Triton  X-100 in CSK buffer to remove membranes and soluble proteins  before formaldehyde fixation, incubated with DNase I, and extracted with 0.25 M ammonium sulfate. The cell preparations  were immunostained with protein 4.1 antibody 24-2 (A, C, E, and  G) and with B4A11 (B, D, F, and H), a monoclonal antibody  against a nuclear matrix protein that displays an intense nuclear  speckle pattern at interphase but is not detectable at mitosis (Blencowe et al., 1994). Micrograph pairs show the fluorescent pattern  with anti–24-2 (left) and B4A11 (right) of the same fields. Cell cycle stages were also confirmed by viewing cells using phase contrast microscopy (not shown). (A) In the center, a mitotic cell  (M) showed staining of the mitotic spindle with particularly  strong staining of the spindle poles. The mitotic cell is surrounded  by interphase cells. Note that at opposite sides of an interphase  nucleus (I), immunostained centrosomes (small spots) are visible.  The inset shows the mitotic spindle of another cell intensely immunolabeled by anti–10-1. (B–F) Epitopes for B4A11 have disappeared in mitotic cells, but a strong speckled staining pattern is  present in interphase nuclei. (C) In the center, a cell in anaphase  retained a high degree of 4.1 staining in the area of the condensed  chromosomes. (E) As the cells approached telophase and cytokinesis, the bridge between the intensely stained perichromosomal  regions became visible by antibody deposition. As the daughter  cells separated further apart (G), bright 4.1 staining appeared at  the midbody (arrow). In the companion B4A11 fields, diffuse  staining began to condense into a more focal pattern, foreshadowing the appearance of the prominent B4A11 speckles characteristic of interphase cells. Bar, 5 μm.
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Figure 9: Dynamic redistribution of protein 4.1 antigens during the cell cycle. CaSki cells were permeabilized with 0.5% Triton X-100 in CSK buffer to remove membranes and soluble proteins before formaldehyde fixation, incubated with DNase I, and extracted with 0.25 M ammonium sulfate. The cell preparations were immunostained with protein 4.1 antibody 24-2 (A, C, E, and G) and with B4A11 (B, D, F, and H), a monoclonal antibody against a nuclear matrix protein that displays an intense nuclear speckle pattern at interphase but is not detectable at mitosis (Blencowe et al., 1994). Micrograph pairs show the fluorescent pattern with anti–24-2 (left) and B4A11 (right) of the same fields. Cell cycle stages were also confirmed by viewing cells using phase contrast microscopy (not shown). (A) In the center, a mitotic cell (M) showed staining of the mitotic spindle with particularly strong staining of the spindle poles. The mitotic cell is surrounded by interphase cells. Note that at opposite sides of an interphase nucleus (I), immunostained centrosomes (small spots) are visible. The inset shows the mitotic spindle of another cell intensely immunolabeled by anti–10-1. (B–F) Epitopes for B4A11 have disappeared in mitotic cells, but a strong speckled staining pattern is present in interphase nuclei. (C) In the center, a cell in anaphase retained a high degree of 4.1 staining in the area of the condensed chromosomes. (E) As the cells approached telophase and cytokinesis, the bridge between the intensely stained perichromosomal regions became visible by antibody deposition. As the daughter cells separated further apart (G), bright 4.1 staining appeared at the midbody (arrow). In the companion B4A11 fields, diffuse staining began to condense into a more focal pattern, foreshadowing the appearance of the prominent B4A11 speckles characteristic of interphase cells. Bar, 5 μm.
Mentions: Using three different classical fixation techniques (paraformaldehyde, methanol, or acetone), prominent punctate staining in the nuclear area of WI38 fibroblasts was consistently observed by indirect immunofluorescence with each antibody. Some antibodies also generated a more diffuse nuclear staining pattern and several of the antibodies produced cytoplasmic staining (Fig. 3). Parallel samples incubated with control IgG did not produce nuclear immunofluorescence with any of these fixation methods (Fig. 3). Fibroblasts were also probed with preparations of 24-2 or 24-3 IgGs directly labeled with either of two different fluorophores; these cells displayed immunofluorescent patterns similar to those obtained by indirect methods (data not shown). Nuclear immunofluorescent staining was also observed when another human fibroblast line (HCA), a transformed epithelial human line (CaSki; see Fig. 9) and a transformed murine fibroblast line (3T3; see Fig. 8) were probed with the 4.1 antibody panel.

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.

Show MeSH
Related in: MedlinePlus