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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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Analysis of fibroblast  nuclear matrix 4.1 protein by  Western blot and 4.1 mRNA by  reverse transcriptase–PCR. (A,  lanes 1–6) Nuclear matrix proteins isolated from 3 × 106 WI38  nuclei were separated by SDSPAGE, transferred to nitrocellulose, and incubated with the  following 4.1 antibodies: lane 1,  RBC 80-kD 4.1; lane 2, N-2; lane  3, 10-1; lane 4, 24-2; lane 5, 24-3;  lane 6, control IgG. To test the  relative separation of nuclear  matrix proteins from soluble cytoplasmic proteins, a cytoplasmic  fraction from 2 × 105 cells was  electrophoresed in parallel with  a sample of nuclear matrix from  6 × 105 cells derived from the  same fibroblast preparation and  probed after transfer with an antibody against the cytoplasmic protein β-tubulin. While β-tubulin was abundant in the cytoplasmic fraction (lane 7), no protein band with a similar migration could be detected in the nuclear matrix fraction (lane 8) even after long exposure  times. An antibody against lamin B used to probe fibroblast nuclear matrix proteins produced a band at the appropriate position, verifying the presence of a predicted nuclear matrix protein (lane 9). (B) PCR analysis of protein 4.1 mRNA containing AUG-1 in WI38 fibroblasts. The products of reverse transcriptase–PCR using primers encompassing AUG-1 were analyzed on a polyacrylamide gel. The  following cDNAs were amplified: lane 1, WI38 cells; lane 2, human reticulocytes; lane 3, no DNA. The sizes of molecular weight standards are: 1353, 1078, 872, 603, 310, 234, and 194 bp.
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Figure 7: Analysis of fibroblast nuclear matrix 4.1 protein by Western blot and 4.1 mRNA by reverse transcriptase–PCR. (A, lanes 1–6) Nuclear matrix proteins isolated from 3 × 106 WI38 nuclei were separated by SDSPAGE, transferred to nitrocellulose, and incubated with the following 4.1 antibodies: lane 1, RBC 80-kD 4.1; lane 2, N-2; lane 3, 10-1; lane 4, 24-2; lane 5, 24-3; lane 6, control IgG. To test the relative separation of nuclear matrix proteins from soluble cytoplasmic proteins, a cytoplasmic fraction from 2 × 105 cells was electrophoresed in parallel with a sample of nuclear matrix from 6 × 105 cells derived from the same fibroblast preparation and probed after transfer with an antibody against the cytoplasmic protein β-tubulin. While β-tubulin was abundant in the cytoplasmic fraction (lane 7), no protein band with a similar migration could be detected in the nuclear matrix fraction (lane 8) even after long exposure times. An antibody against lamin B used to probe fibroblast nuclear matrix proteins produced a band at the appropriate position, verifying the presence of a predicted nuclear matrix protein (lane 9). (B) PCR analysis of protein 4.1 mRNA containing AUG-1 in WI38 fibroblasts. The products of reverse transcriptase–PCR using primers encompassing AUG-1 were analyzed on a polyacrylamide gel. The following cDNAs were amplified: lane 1, WI38 cells; lane 2, human reticulocytes; lane 3, no DNA. The sizes of molecular weight standards are: 1353, 1078, 872, 603, 310, 234, and 194 bp.

Mentions: To analyze nuclear-associated 4.1 isoforms, nuclear matrix proteins isolated from purified fibroblast nuclei were separated on SDS-PAGE and transferred to nitrocellulose, and identical gel lanes were probed with each of the 4.1 antibodies. A number of protein bands in the matrix preparation were reactive with multiple 4.1 IgGs. (Fig. 7 A, lanes 1–5). Prominent clusters of bands migrating with molecular masses in the range of ∼55 and 80–100 kD were detected. Other less abundant bands also reacted with antibodies against several different protein 4.1 epitopes: ∼65 kD (antibodies 24-2, 24-3) and ∼120–150 kD (antibodies 10-1, 24-3, 4.1). No reaction with the nuclear matrix proteins occurred when control rabbit IgG was used as a probe (Fig. 7 A, lane 6). Thus multiple isoforms of 4.1 are retained in the most insoluble fraction of nuclear proteins.


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)

Analysis of fibroblast  nuclear matrix 4.1 protein by  Western blot and 4.1 mRNA by  reverse transcriptase–PCR. (A,  lanes 1–6) Nuclear matrix proteins isolated from 3 × 106 WI38  nuclei were separated by SDSPAGE, transferred to nitrocellulose, and incubated with the  following 4.1 antibodies: lane 1,  RBC 80-kD 4.1; lane 2, N-2; lane  3, 10-1; lane 4, 24-2; lane 5, 24-3;  lane 6, control IgG. To test the  relative separation of nuclear  matrix proteins from soluble cytoplasmic proteins, a cytoplasmic  fraction from 2 × 105 cells was  electrophoresed in parallel with  a sample of nuclear matrix from  6 × 105 cells derived from the  same fibroblast preparation and  probed after transfer with an antibody against the cytoplasmic protein β-tubulin. While β-tubulin was abundant in the cytoplasmic fraction (lane 7), no protein band with a similar migration could be detected in the nuclear matrix fraction (lane 8) even after long exposure  times. An antibody against lamin B used to probe fibroblast nuclear matrix proteins produced a band at the appropriate position, verifying the presence of a predicted nuclear matrix protein (lane 9). (B) PCR analysis of protein 4.1 mRNA containing AUG-1 in WI38 fibroblasts. The products of reverse transcriptase–PCR using primers encompassing AUG-1 were analyzed on a polyacrylamide gel. The  following cDNAs were amplified: lane 1, WI38 cells; lane 2, human reticulocytes; lane 3, no DNA. The sizes of molecular weight standards are: 1353, 1078, 872, 603, 310, 234, and 194 bp.
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Figure 7: Analysis of fibroblast nuclear matrix 4.1 protein by Western blot and 4.1 mRNA by reverse transcriptase–PCR. (A, lanes 1–6) Nuclear matrix proteins isolated from 3 × 106 WI38 nuclei were separated by SDSPAGE, transferred to nitrocellulose, and incubated with the following 4.1 antibodies: lane 1, RBC 80-kD 4.1; lane 2, N-2; lane 3, 10-1; lane 4, 24-2; lane 5, 24-3; lane 6, control IgG. To test the relative separation of nuclear matrix proteins from soluble cytoplasmic proteins, a cytoplasmic fraction from 2 × 105 cells was electrophoresed in parallel with a sample of nuclear matrix from 6 × 105 cells derived from the same fibroblast preparation and probed after transfer with an antibody against the cytoplasmic protein β-tubulin. While β-tubulin was abundant in the cytoplasmic fraction (lane 7), no protein band with a similar migration could be detected in the nuclear matrix fraction (lane 8) even after long exposure times. An antibody against lamin B used to probe fibroblast nuclear matrix proteins produced a band at the appropriate position, verifying the presence of a predicted nuclear matrix protein (lane 9). (B) PCR analysis of protein 4.1 mRNA containing AUG-1 in WI38 fibroblasts. The products of reverse transcriptase–PCR using primers encompassing AUG-1 were analyzed on a polyacrylamide gel. The following cDNAs were amplified: lane 1, WI38 cells; lane 2, human reticulocytes; lane 3, no DNA. The sizes of molecular weight standards are: 1353, 1078, 872, 603, 310, 234, and 194 bp.
Mentions: To analyze nuclear-associated 4.1 isoforms, nuclear matrix proteins isolated from purified fibroblast nuclei were separated on SDS-PAGE and transferred to nitrocellulose, and identical gel lanes were probed with each of the 4.1 antibodies. A number of protein bands in the matrix preparation were reactive with multiple 4.1 IgGs. (Fig. 7 A, lanes 1–5). Prominent clusters of bands migrating with molecular masses in the range of ∼55 and 80–100 kD were detected. Other less abundant bands also reacted with antibodies against several different protein 4.1 epitopes: ∼65 kD (antibodies 24-2, 24-3) and ∼120–150 kD (antibodies 10-1, 24-3, 4.1). No reaction with the nuclear matrix proteins occurred when control rabbit IgG was used as a probe (Fig. 7 A, lane 6). Thus multiple isoforms of 4.1 are retained in the most insoluble fraction of nuclear proteins.

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