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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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Characterization of antibodies directed against red cell 80-kD 4.1 and 4.1 peptide domains. (A) Human red cell membrane  fractions (Conboy et al., 1993) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with the following IgGs:  lane 1, anti-RBC 4.1; lane 2, anti–N-2; lane 3, anti–10-1; lane 4, anti–24-2; lane 5, anti–24-3; lane 6, control IgG. Protein 4.1 in red cells migrates at ∼80 kD. Although this is the predominant 4.1 isoform in red cells, a faint band at ∼135 kD detected by anti–N-2 (lane 2) is also  detected by other 4.1 IgGs when increased amounts of red cell membrane preparations are applied to gels (Conboy et al., 1991). (B) Human red cells were fixed by methanol and probed by indirect immunofluorescence using primary antibodies as indicated and FITCconjugated secondary antibodies. The faint staining by anti–N-2 most likely reflects the small amount of ∼135-kD 4.1 also detected by  Western blot analysis of red cell membranes. Bar, 10 μm.
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Figure 2: Characterization of antibodies directed against red cell 80-kD 4.1 and 4.1 peptide domains. (A) Human red cell membrane fractions (Conboy et al., 1993) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with the following IgGs: lane 1, anti-RBC 4.1; lane 2, anti–N-2; lane 3, anti–10-1; lane 4, anti–24-2; lane 5, anti–24-3; lane 6, control IgG. Protein 4.1 in red cells migrates at ∼80 kD. Although this is the predominant 4.1 isoform in red cells, a faint band at ∼135 kD detected by anti–N-2 (lane 2) is also detected by other 4.1 IgGs when increased amounts of red cell membrane preparations are applied to gels (Conboy et al., 1991). (B) Human red cells were fixed by methanol and probed by indirect immunofluorescence using primary antibodies as indicated and FITCconjugated secondary antibodies. The faint staining by anti–N-2 most likely reflects the small amount of ∼135-kD 4.1 also detected by Western blot analysis of red cell membranes. Bar, 10 μm.

Mentions: To detect 4.1 epitopes by immunofluorescence microscopy, we used a panel of affinity-purified IgGs. As depicted by the map of multiple alternative splicing pathways in Fig. 1 A, protein 4.1 isoforms contain varying patterns of exonic inclusion and exclusion. Using exonspecific antibodies allows detection of 4.1 isoforms having the peptide encoded by that particular exon. Our antibody panel included antibody 10-1, recognizing sequence encoded by exon 16; antibody 24-2, specific for peptide encoded by exon 19; antibody 24-3, recognizing epitope encoded by exon 21; antibody N-2, recognizing the NH2-terminal 209 amino acids translated from the AUG-1 start site (Fig 1, B and C); and anti-RBC 80-kD, against the 80-kD red cell 4.1 isoform purified from human RBC (Fig. 1 B). We established the specificity of our antibody panel for protein 4.1 by showing that IgGs 10-1, 24-2, 24-3, and anti-RBC 80-kD 4.1 each reacted with red cell 80-kD 4.1 in Western blot analysis (Fig. 2 A) and with protein 4.1 in the plasma membrane of human red cells using immunofluorescent detection (Fig. 2 B). Furthermore, immunofluorescent signals were coincident when human red blood cells were probed with 24-2 and 24-3 directly labeled with two different fluorophores (see Fig. 4).


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)

Characterization of antibodies directed against red cell 80-kD 4.1 and 4.1 peptide domains. (A) Human red cell membrane  fractions (Conboy et al., 1993) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with the following IgGs:  lane 1, anti-RBC 4.1; lane 2, anti–N-2; lane 3, anti–10-1; lane 4, anti–24-2; lane 5, anti–24-3; lane 6, control IgG. Protein 4.1 in red cells migrates at ∼80 kD. Although this is the predominant 4.1 isoform in red cells, a faint band at ∼135 kD detected by anti–N-2 (lane 2) is also  detected by other 4.1 IgGs when increased amounts of red cell membrane preparations are applied to gels (Conboy et al., 1991). (B) Human red cells were fixed by methanol and probed by indirect immunofluorescence using primary antibodies as indicated and FITCconjugated secondary antibodies. The faint staining by anti–N-2 most likely reflects the small amount of ∼135-kD 4.1 also detected by  Western blot analysis of red cell membranes. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139783&req=5

Figure 2: Characterization of antibodies directed against red cell 80-kD 4.1 and 4.1 peptide domains. (A) Human red cell membrane fractions (Conboy et al., 1993) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with the following IgGs: lane 1, anti-RBC 4.1; lane 2, anti–N-2; lane 3, anti–10-1; lane 4, anti–24-2; lane 5, anti–24-3; lane 6, control IgG. Protein 4.1 in red cells migrates at ∼80 kD. Although this is the predominant 4.1 isoform in red cells, a faint band at ∼135 kD detected by anti–N-2 (lane 2) is also detected by other 4.1 IgGs when increased amounts of red cell membrane preparations are applied to gels (Conboy et al., 1991). (B) Human red cells were fixed by methanol and probed by indirect immunofluorescence using primary antibodies as indicated and FITCconjugated secondary antibodies. The faint staining by anti–N-2 most likely reflects the small amount of ∼135-kD 4.1 also detected by Western blot analysis of red cell membranes. Bar, 10 μm.
Mentions: To detect 4.1 epitopes by immunofluorescence microscopy, we used a panel of affinity-purified IgGs. As depicted by the map of multiple alternative splicing pathways in Fig. 1 A, protein 4.1 isoforms contain varying patterns of exonic inclusion and exclusion. Using exonspecific antibodies allows detection of 4.1 isoforms having the peptide encoded by that particular exon. Our antibody panel included antibody 10-1, recognizing sequence encoded by exon 16; antibody 24-2, specific for peptide encoded by exon 19; antibody 24-3, recognizing epitope encoded by exon 21; antibody N-2, recognizing the NH2-terminal 209 amino acids translated from the AUG-1 start site (Fig 1, B and C); and anti-RBC 80-kD, against the 80-kD red cell 4.1 isoform purified from human RBC (Fig. 1 B). We established the specificity of our antibody panel for protein 4.1 by showing that IgGs 10-1, 24-2, 24-3, and anti-RBC 80-kD 4.1 each reacted with red cell 80-kD 4.1 in Western blot analysis (Fig. 2 A) and with protein 4.1 in the plasma membrane of human red cells using immunofluorescent detection (Fig. 2 B). Furthermore, immunofluorescent signals were coincident when human red blood cells were probed with 24-2 and 24-3 directly labeled with two different fluorophores (see Fig. 4).

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