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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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Detection of nuclear epitopes of protein 4.1 by immunofluorescent light microscopy in WI38 cells. Cultured WI38 human fibroblasts were fixed in acetone for probing with anti-RBC 80-kD 4.1 and N-2, in methanol for probing with anti–24-2, or in formaldehyde for probing with anti–10-1 and anti–24-3, followed by incubation with a FITC-labeled secondary antibody. Punctate nuclear signals, detected with all the protein 4.1 antibodies, were particularly prominent with anti-RBC 80-kD 4.1 and anti–24-2. Localization of  FITC signals within the nuclei of all cells was confirmed by comparison to DAPI fluorescence of the cells with 4.1 fluorescence (not  shown). Antibodies N-2, 10-1, and 24-3 also produced considerable cytoplasmic staining. Staining patterns were in large part independent  of the fixation method. Controls showed no fluorescent patterns when imaged under the same conditions as experimental samples. Bar, 10 μm.
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Figure 3: Detection of nuclear epitopes of protein 4.1 by immunofluorescent light microscopy in WI38 cells. Cultured WI38 human fibroblasts were fixed in acetone for probing with anti-RBC 80-kD 4.1 and N-2, in methanol for probing with anti–24-2, or in formaldehyde for probing with anti–10-1 and anti–24-3, followed by incubation with a FITC-labeled secondary antibody. Punctate nuclear signals, detected with all the protein 4.1 antibodies, were particularly prominent with anti-RBC 80-kD 4.1 and anti–24-2. Localization of FITC signals within the nuclei of all cells was confirmed by comparison to DAPI fluorescence of the cells with 4.1 fluorescence (not shown). Antibodies N-2, 10-1, and 24-3 also produced considerable cytoplasmic staining. Staining patterns were in large part independent of the fixation method. Controls showed no fluorescent patterns when imaged under the same conditions as experimental samples. Bar, 10 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)

Detection of nuclear epitopes of protein 4.1 by immunofluorescent light microscopy in WI38 cells. Cultured WI38 human fibroblasts were fixed in acetone for probing with anti-RBC 80-kD 4.1 and N-2, in methanol for probing with anti–24-2, or in formaldehyde for probing with anti–10-1 and anti–24-3, followed by incubation with a FITC-labeled secondary antibody. Punctate nuclear signals, detected with all the protein 4.1 antibodies, were particularly prominent with anti-RBC 80-kD 4.1 and anti–24-2. Localization of  FITC signals within the nuclei of all cells was confirmed by comparison to DAPI fluorescence of the cells with 4.1 fluorescence (not  shown). Antibodies N-2, 10-1, and 24-3 also produced considerable cytoplasmic staining. Staining patterns were in large part independent  of the fixation method. Controls showed no fluorescent patterns when imaged under the same conditions as experimental samples. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139783&req=5

Figure 3: Detection of nuclear epitopes of protein 4.1 by immunofluorescent light microscopy in WI38 cells. Cultured WI38 human fibroblasts were fixed in acetone for probing with anti-RBC 80-kD 4.1 and N-2, in methanol for probing with anti–24-2, or in formaldehyde for probing with anti–10-1 and anti–24-3, followed by incubation with a FITC-labeled secondary antibody. Punctate nuclear signals, detected with all the protein 4.1 antibodies, were particularly prominent with anti-RBC 80-kD 4.1 and anti–24-2. Localization of FITC signals within the nuclei of all cells was confirmed by comparison to DAPI fluorescence of the cells with 4.1 fluorescence (not shown). Antibodies N-2, 10-1, and 24-3 also produced considerable cytoplasmic staining. Staining patterns were in large part independent of the fixation method. Controls showed no fluorescent patterns when imaged under the same conditions as experimental samples. Bar, 10 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