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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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Protein 4.1 peptide  domains used to generate antibodies. (A) A schematic diagram of protein 4.1 mRNAs  displaying multiple combinations of splicing pathways  possible among 4.1 alternative exons. In this format, exons are coded as follows:  solid bars, constitutive;  shaded bars, alternative;  open bars, noncoding. The  arrows on top indicate the  positions of alternative translation initiation sites, AUG-1  and AUG-2. The figure is derived from Conboy et al.  (1991). (B) Examples of protein 4.1 isoforms derived  from different translation initiation sites. The 80-kD prototypical red cell isoform is produced from AUG-2 and can be present in nucleated and nonnucleated cells.  Chymotryptic fragments of this isoform include a 30-kD membrane binding domain, a 16-kD domain, a 10-kD spectrin and actin binding domain, and the 22–24-kD domain (Leto and Marchesi, 1984). Higher molecular mass 4.1 isoforms, present in nucleated cells, use  AUG-1 to generate an additional 209–amino acid “NH2-terminal extension” (N-term). (C) Synthetic peptides derived from the 4.1  amino acid sequence at the positions indicated by the arrows were used to immunize rabbits. IgGs were prepared from N-2, 10-1, 24-2, and  24-3 sera by affinity purification using the homologous peptide. The sequences for the peptides are given in the Methods section.
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Figure 1: Protein 4.1 peptide domains used to generate antibodies. (A) A schematic diagram of protein 4.1 mRNAs displaying multiple combinations of splicing pathways possible among 4.1 alternative exons. In this format, exons are coded as follows: solid bars, constitutive; shaded bars, alternative; open bars, noncoding. The arrows on top indicate the positions of alternative translation initiation sites, AUG-1 and AUG-2. The figure is derived from Conboy et al. (1991). (B) Examples of protein 4.1 isoforms derived from different translation initiation sites. The 80-kD prototypical red cell isoform is produced from AUG-2 and can be present in nucleated and nonnucleated cells. Chymotryptic fragments of this isoform include a 30-kD membrane binding domain, a 16-kD domain, a 10-kD spectrin and actin binding domain, and the 22–24-kD domain (Leto and Marchesi, 1984). Higher molecular mass 4.1 isoforms, present in nucleated cells, use AUG-1 to generate an additional 209–amino acid “NH2-terminal extension” (N-term). (C) Synthetic peptides derived from the 4.1 amino acid sequence at the positions indicated by the arrows were used to immunize rabbits. IgGs were prepared from N-2, 10-1, 24-2, and 24-3 sera by affinity purification using the homologous peptide. The sequences for the peptides are given in the Methods section.

Mentions: Rabbits were immunized with 80-kD protein 4.1 purified from human RBC or with synthetic peptides comprised of sequences encoded by either exon 16 (peptide 10-1), exon 19 (peptide 24-2), or exon 21 (peptide 24-3) coupled to thyroglobulin (see Fig. 1). Sequences of the peptide antigens were: 10-1, KKRERLDGENIYIRHSNLMLEC; 24-2, TDDNSGDLDPGVLLTAQTITSETPSSTTTTQITKC; or 24-3, HPDMSVTKVVVH QETEIADEC. In addition, the sequence encoding peptide N-2, containing 209 amino acids following the AUG-1 start site of translation of protein 4.1 (Conboy et al., 1991) (Fig. 1), was fused to glutathione-S-transferase (GST). The construct was overexpressed, purified by glutathione agarose affinity chromatography, and used for immunization. Immune IgGs were affinity purified against the immunizing 4.1 peptide or RBC 80-kD 4.1 and analyzed to confirm no cross-reaction with either thyroglobulin or the GST moiety. Antibodies 10-1 and 24-2 have been used by us in other studies (Chasis et al., 1993; Conboy et al., 1993; Schischmanoff et al., 1995; Chasis et al., 1996). For direct labeling, IgGs (0.4 μg/ml) were conjugated to rhodamine or FITC fluorophores and then extensively dialyzed according to the instructions provided by Molecular Probes.


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)

Protein 4.1 peptide  domains used to generate antibodies. (A) A schematic diagram of protein 4.1 mRNAs  displaying multiple combinations of splicing pathways  possible among 4.1 alternative exons. In this format, exons are coded as follows:  solid bars, constitutive;  shaded bars, alternative;  open bars, noncoding. The  arrows on top indicate the  positions of alternative translation initiation sites, AUG-1  and AUG-2. The figure is derived from Conboy et al.  (1991). (B) Examples of protein 4.1 isoforms derived  from different translation initiation sites. The 80-kD prototypical red cell isoform is produced from AUG-2 and can be present in nucleated and nonnucleated cells.  Chymotryptic fragments of this isoform include a 30-kD membrane binding domain, a 16-kD domain, a 10-kD spectrin and actin binding domain, and the 22–24-kD domain (Leto and Marchesi, 1984). Higher molecular mass 4.1 isoforms, present in nucleated cells, use  AUG-1 to generate an additional 209–amino acid “NH2-terminal extension” (N-term). (C) Synthetic peptides derived from the 4.1  amino acid sequence at the positions indicated by the arrows were used to immunize rabbits. IgGs were prepared from N-2, 10-1, 24-2, and  24-3 sera by affinity purification using the homologous peptide. The sequences for the peptides are given in the Methods section.
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Related In: Results  -  Collection

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Figure 1: Protein 4.1 peptide domains used to generate antibodies. (A) A schematic diagram of protein 4.1 mRNAs displaying multiple combinations of splicing pathways possible among 4.1 alternative exons. In this format, exons are coded as follows: solid bars, constitutive; shaded bars, alternative; open bars, noncoding. The arrows on top indicate the positions of alternative translation initiation sites, AUG-1 and AUG-2. The figure is derived from Conboy et al. (1991). (B) Examples of protein 4.1 isoforms derived from different translation initiation sites. The 80-kD prototypical red cell isoform is produced from AUG-2 and can be present in nucleated and nonnucleated cells. Chymotryptic fragments of this isoform include a 30-kD membrane binding domain, a 16-kD domain, a 10-kD spectrin and actin binding domain, and the 22–24-kD domain (Leto and Marchesi, 1984). Higher molecular mass 4.1 isoforms, present in nucleated cells, use AUG-1 to generate an additional 209–amino acid “NH2-terminal extension” (N-term). (C) Synthetic peptides derived from the 4.1 amino acid sequence at the positions indicated by the arrows were used to immunize rabbits. IgGs were prepared from N-2, 10-1, 24-2, and 24-3 sera by affinity purification using the homologous peptide. The sequences for the peptides are given in the Methods section.
Mentions: Rabbits were immunized with 80-kD protein 4.1 purified from human RBC or with synthetic peptides comprised of sequences encoded by either exon 16 (peptide 10-1), exon 19 (peptide 24-2), or exon 21 (peptide 24-3) coupled to thyroglobulin (see Fig. 1). Sequences of the peptide antigens were: 10-1, KKRERLDGENIYIRHSNLMLEC; 24-2, TDDNSGDLDPGVLLTAQTITSETPSSTTTTQITKC; or 24-3, HPDMSVTKVVVH QETEIADEC. In addition, the sequence encoding peptide N-2, containing 209 amino acids following the AUG-1 start site of translation of protein 4.1 (Conboy et al., 1991) (Fig. 1), was fused to glutathione-S-transferase (GST). The construct was overexpressed, purified by glutathione agarose affinity chromatography, and used for immunization. Immune IgGs were affinity purified against the immunizing 4.1 peptide or RBC 80-kD 4.1 and analyzed to confirm no cross-reaction with either thyroglobulin or the GST moiety. Antibodies 10-1 and 24-2 have been used by us in other studies (Chasis et al., 1993; Conboy et al., 1993; Schischmanoff et al., 1995; Chasis et al., 1996). For direct labeling, IgGs (0.4 μg/ml) were conjugated to rhodamine or FITC fluorophores and then extensively dialyzed according to the instructions provided by Molecular Probes.

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