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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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Expression of epitope-tagged 4.1 in nuclei after transient transfection. A construct was engineered to encode the sequences  for red cell 80-kD protein 4.1 fused to an epitope tag derived from SV-40 large T antigen. (A) The construct was bacterially expressed,  isolated, and then analyzed by Western blotting to confirm the presence of both 4.1 and SV-40 tag epitopes. Both 24-2 IgG (lane 1) and  KT3 antibody (against the epitope tag; lane 2) recognized a protein with the same apparent molecular mass. The KT3 antibody did not  recognize epitopes in a whole cell lysate of 3T3 cells (lane 3). (B) Murine fibroblast 3T3 cells, probed with anti-RBC 80-kD 4.1, displayed punctate nuclear immunofluorescent signals. (C and C′) 3T3 cells, transiently transfected with pSV40NeoCMV containing sequences encoding epitope-tagged RBC 80-kD 4.1, strongly expressed epitope-tagged protein localized in nuclei, which was detected by  indirect immunofluorescence using KT3 antibody. (D and D′) After parallel transient transfection of 3T3 cells with pSV40NeoCMV  without a construct inserted, there was no immunofluorescent staining with KT3 antibody. Bar: (B and B′) 12 μm; (C–D′) 20 μm.
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Figure 8: Expression of epitope-tagged 4.1 in nuclei after transient transfection. A construct was engineered to encode the sequences for red cell 80-kD protein 4.1 fused to an epitope tag derived from SV-40 large T antigen. (A) The construct was bacterially expressed, isolated, and then analyzed by Western blotting to confirm the presence of both 4.1 and SV-40 tag epitopes. Both 24-2 IgG (lane 1) and KT3 antibody (against the epitope tag; lane 2) recognized a protein with the same apparent molecular mass. The KT3 antibody did not recognize epitopes in a whole cell lysate of 3T3 cells (lane 3). (B) Murine fibroblast 3T3 cells, probed with anti-RBC 80-kD 4.1, displayed punctate nuclear immunofluorescent signals. (C and C′) 3T3 cells, transiently transfected with pSV40NeoCMV containing sequences encoding epitope-tagged RBC 80-kD 4.1, strongly expressed epitope-tagged protein localized in nuclei, which was detected by indirect immunofluorescence using KT3 antibody. (D and D′) After parallel transient transfection of 3T3 cells with pSV40NeoCMV without a construct inserted, there was no immunofluorescent staining with KT3 antibody. Bar: (B and B′) 12 μm; (C–D′) 20 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)

Expression of epitope-tagged 4.1 in nuclei after transient transfection. A construct was engineered to encode the sequences  for red cell 80-kD protein 4.1 fused to an epitope tag derived from SV-40 large T antigen. (A) The construct was bacterially expressed,  isolated, and then analyzed by Western blotting to confirm the presence of both 4.1 and SV-40 tag epitopes. Both 24-2 IgG (lane 1) and  KT3 antibody (against the epitope tag; lane 2) recognized a protein with the same apparent molecular mass. The KT3 antibody did not  recognize epitopes in a whole cell lysate of 3T3 cells (lane 3). (B) Murine fibroblast 3T3 cells, probed with anti-RBC 80-kD 4.1, displayed punctate nuclear immunofluorescent signals. (C and C′) 3T3 cells, transiently transfected with pSV40NeoCMV containing sequences encoding epitope-tagged RBC 80-kD 4.1, strongly expressed epitope-tagged protein localized in nuclei, which was detected by  indirect immunofluorescence using KT3 antibody. (D and D′) After parallel transient transfection of 3T3 cells with pSV40NeoCMV  without a construct inserted, there was no immunofluorescent staining with KT3 antibody. Bar: (B and B′) 12 μm; (C–D′) 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139783&req=5

Figure 8: Expression of epitope-tagged 4.1 in nuclei after transient transfection. A construct was engineered to encode the sequences for red cell 80-kD protein 4.1 fused to an epitope tag derived from SV-40 large T antigen. (A) The construct was bacterially expressed, isolated, and then analyzed by Western blotting to confirm the presence of both 4.1 and SV-40 tag epitopes. Both 24-2 IgG (lane 1) and KT3 antibody (against the epitope tag; lane 2) recognized a protein with the same apparent molecular mass. The KT3 antibody did not recognize epitopes in a whole cell lysate of 3T3 cells (lane 3). (B) Murine fibroblast 3T3 cells, probed with anti-RBC 80-kD 4.1, displayed punctate nuclear immunofluorescent signals. (C and C′) 3T3 cells, transiently transfected with pSV40NeoCMV containing sequences encoding epitope-tagged RBC 80-kD 4.1, strongly expressed epitope-tagged protein localized in nuclei, which was detected by indirect immunofluorescence using KT3 antibody. (D and D′) After parallel transient transfection of 3T3 cells with pSV40NeoCMV without a construct inserted, there was no immunofluorescent staining with KT3 antibody. Bar: (B and B′) 12 μm; (C–D′) 20 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