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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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Distribution of protein 4.1 epitopes in the interphase nucleus of human fibroblasts visualized by immunofluorescence. Location of the nucleus was confirmed in all cases by DAPI staining (not shown). (A and B) Double-label confocal microscopy of cells  stained by protein 4.1 antibody 24-2 (green) and (A) antibody against nuclear pores (red) or (B) lamin B (red). The majority of nuclear  protein 4.1 foci are interior to the periphery of the nucleus as demarcated by immunofluorescence of pores within nuclear membrane  and by its subadjacent network of lamin B fibers. (Additional lamin B sites in the nuclear interior have been reported [Bridger et al.,  1993; Moir et al., 1994]). Similar results were obtained by imaging epitopes for protein 4.1 antibodies 24-3, 10-1, and N-2 relative to antipore  and antilamin epitopes. (C) Three-dimensional image analysis of nuclear epitopes probed with anti-RBC 80-kD 4.1 showing a 0.3-μm  horizontal optical midsection of an immunofluorescent cell with a line indicating the position of the vertical reconstruction depicted in  C′. Since optical resolution in the z-axis (vertical) is less than in the x-y (horizontal) axis, the reconstructed image is less sharp; the dome  shape of the top of the cell and the flat bottom plane of the coverslip are somewhat obscured by the coincidence of out of focus light. (A  cartoon depicting the approximate shape of the vertical cross-section through the nucleus is presented in the bottom panel.) Thus, it is apparent that epitopes for protein 4.1 lie in numerous planes within the volume of the cell. Similar results were obtained in three-dimensional reconstructions using the 24-2, 24-3, and 10-1 antibodies as probes. (D and D′) Double-label confocal microscopy of a fibroblast  using anti–24-2 (green) and anti-NuMA (red). In a horizontal plane through the midsection of the cell (D), protein 4.1 foci appeared  within the NuMA-stained nuclear interior. (D′) Three-dimensional reconstructions were made of three different vertical planes from  this NuMA:24-2 double-labeled cell. The flattened plane of the coverslip appears at the top of the vertical reconstructed images. The  conclusion that 4.1 epitopes localized at multiple planes throughout the volume of the nucleus is entirely consistent with the observations  obtained in images A, B, and C. The same conclusion was reached when cells were probed with antibodies against NuMA and RBC 80-kD  protein 4.1. (E and F) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against PCNA (red).  Optical sections through the fibroblast nucleus show that 24-2 (green) and PCNA (red) epitopes coincided (yellow) in many areas except  in the vicinities of the nucleoli, which either are relatively devoid of any signal or sometimes contained a clustering of 24-2 (green) signals. Both PCNA and 24-2 epitopes resided in multiple planes throughout the nucleus (not shown). (G and H) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against SC-35 (red). Optical sections are in the plane of the cell containing SC-35 domains; most SC-35 domains in fibroblasts are located in a single plane (Carter et al., 1993). It is apparent that the vast  majority of the SC-35 domains contained areas of coincident staining with 24-2 (seen as yellow coloration), most often at the periphery  of the SC-35 domains. In optical sections above and below SC-35, additional 24-2 signals are observed (not shown), consistent with the  conclusions from the localization data presented in A–F. Bars, 3.5 μm (8 μm in the vertical dimension [C′ and D′]).
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Figure 5: Distribution of protein 4.1 epitopes in the interphase nucleus of human fibroblasts visualized by immunofluorescence. Location of the nucleus was confirmed in all cases by DAPI staining (not shown). (A and B) Double-label confocal microscopy of cells stained by protein 4.1 antibody 24-2 (green) and (A) antibody against nuclear pores (red) or (B) lamin B (red). The majority of nuclear protein 4.1 foci are interior to the periphery of the nucleus as demarcated by immunofluorescence of pores within nuclear membrane and by its subadjacent network of lamin B fibers. (Additional lamin B sites in the nuclear interior have been reported [Bridger et al., 1993; Moir et al., 1994]). Similar results were obtained by imaging epitopes for protein 4.1 antibodies 24-3, 10-1, and N-2 relative to antipore and antilamin epitopes. (C) Three-dimensional image analysis of nuclear epitopes probed with anti-RBC 80-kD 4.1 showing a 0.3-μm horizontal optical midsection of an immunofluorescent cell with a line indicating the position of the vertical reconstruction depicted in C′. Since optical resolution in the z-axis (vertical) is less than in the x-y (horizontal) axis, the reconstructed image is less sharp; the dome shape of the top of the cell and the flat bottom plane of the coverslip are somewhat obscured by the coincidence of out of focus light. (A cartoon depicting the approximate shape of the vertical cross-section through the nucleus is presented in the bottom panel.) Thus, it is apparent that epitopes for protein 4.1 lie in numerous planes within the volume of the cell. Similar results were obtained in three-dimensional reconstructions using the 24-2, 24-3, and 10-1 antibodies as probes. (D and D′) Double-label confocal microscopy of a fibroblast using anti–24-2 (green) and anti-NuMA (red). In a horizontal plane through the midsection of the cell (D), protein 4.1 foci appeared within the NuMA-stained nuclear interior. (D′) Three-dimensional reconstructions were made of three different vertical planes from this NuMA:24-2 double-labeled cell. The flattened plane of the coverslip appears at the top of the vertical reconstructed images. The conclusion that 4.1 epitopes localized at multiple planes throughout the volume of the nucleus is entirely consistent with the observations obtained in images A, B, and C. The same conclusion was reached when cells were probed with antibodies against NuMA and RBC 80-kD protein 4.1. (E and F) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against PCNA (red). Optical sections through the fibroblast nucleus show that 24-2 (green) and PCNA (red) epitopes coincided (yellow) in many areas except in the vicinities of the nucleoli, which either are relatively devoid of any signal or sometimes contained a clustering of 24-2 (green) signals. Both PCNA and 24-2 epitopes resided in multiple planes throughout the nucleus (not shown). (G and H) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against SC-35 (red). Optical sections are in the plane of the cell containing SC-35 domains; most SC-35 domains in fibroblasts are located in a single plane (Carter et al., 1993). It is apparent that the vast majority of the SC-35 domains contained areas of coincident staining with 24-2 (seen as yellow coloration), most often at the periphery of the SC-35 domains. In optical sections above and below SC-35, additional 24-2 signals are observed (not shown), consistent with the conclusions from the localization data presented in A–F. Bars, 3.5 μm (8 μm in the vertical dimension [C′ and D′]).

Mentions: The nucleus is bounded by two concentric lipid bilayers perforated by nuclear pores and an underlying ring of lamin, a specialized intermediate filament. Nuclear-associated 4.1 immunofluorescent signals could have arisen from cytoplasmic epitopes overlying the nucleus, from nuclear membrane sites, or from interior nuclear locations. In fact, since 80-kD protein 4.1 in mature erythrocytes is a component of the plasma membrane skeleton (Fig. 4), one might predict that 4.1 would localize to the nuclear membrane or even the underlying lamin protein ring. To distinguish among these possibilities, double-label confocal microscopy was performed to localize 4.1 foci relative to epitopes for two well-characterized nuclear proteins, lamin B and the nuclear pore complex. Optical horizontal sections through the center of the nucleus revealed well-separated rhodamine (Fig. 5 A, stain for lamin; Fig. 5 B, stain for pores) and FITC (stain for 4.1) signals, leading to the conclusion that 4.1 epitopes are within the area bounded by nuclear pores embedded in the nuclear membrane and by the underlying lamin protein ring. Furthermore, the smooth circumferential staining of both pores and lamin in the preparations rules out the possibility that internal 4.1 foci are being generated by invaginations of the nuclear membrane.


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)

Distribution of protein 4.1 epitopes in the interphase nucleus of human fibroblasts visualized by immunofluorescence. Location of the nucleus was confirmed in all cases by DAPI staining (not shown). (A and B) Double-label confocal microscopy of cells  stained by protein 4.1 antibody 24-2 (green) and (A) antibody against nuclear pores (red) or (B) lamin B (red). The majority of nuclear  protein 4.1 foci are interior to the periphery of the nucleus as demarcated by immunofluorescence of pores within nuclear membrane  and by its subadjacent network of lamin B fibers. (Additional lamin B sites in the nuclear interior have been reported [Bridger et al.,  1993; Moir et al., 1994]). Similar results were obtained by imaging epitopes for protein 4.1 antibodies 24-3, 10-1, and N-2 relative to antipore  and antilamin epitopes. (C) Three-dimensional image analysis of nuclear epitopes probed with anti-RBC 80-kD 4.1 showing a 0.3-μm  horizontal optical midsection of an immunofluorescent cell with a line indicating the position of the vertical reconstruction depicted in  C′. Since optical resolution in the z-axis (vertical) is less than in the x-y (horizontal) axis, the reconstructed image is less sharp; the dome  shape of the top of the cell and the flat bottom plane of the coverslip are somewhat obscured by the coincidence of out of focus light. (A  cartoon depicting the approximate shape of the vertical cross-section through the nucleus is presented in the bottom panel.) Thus, it is apparent that epitopes for protein 4.1 lie in numerous planes within the volume of the cell. Similar results were obtained in three-dimensional reconstructions using the 24-2, 24-3, and 10-1 antibodies as probes. (D and D′) Double-label confocal microscopy of a fibroblast  using anti–24-2 (green) and anti-NuMA (red). In a horizontal plane through the midsection of the cell (D), protein 4.1 foci appeared  within the NuMA-stained nuclear interior. (D′) Three-dimensional reconstructions were made of three different vertical planes from  this NuMA:24-2 double-labeled cell. The flattened plane of the coverslip appears at the top of the vertical reconstructed images. The  conclusion that 4.1 epitopes localized at multiple planes throughout the volume of the nucleus is entirely consistent with the observations  obtained in images A, B, and C. The same conclusion was reached when cells were probed with antibodies against NuMA and RBC 80-kD  protein 4.1. (E and F) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against PCNA (red).  Optical sections through the fibroblast nucleus show that 24-2 (green) and PCNA (red) epitopes coincided (yellow) in many areas except  in the vicinities of the nucleoli, which either are relatively devoid of any signal or sometimes contained a clustering of 24-2 (green) signals. Both PCNA and 24-2 epitopes resided in multiple planes throughout the nucleus (not shown). (G and H) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against SC-35 (red). Optical sections are in the plane of the cell containing SC-35 domains; most SC-35 domains in fibroblasts are located in a single plane (Carter et al., 1993). It is apparent that the vast  majority of the SC-35 domains contained areas of coincident staining with 24-2 (seen as yellow coloration), most often at the periphery  of the SC-35 domains. In optical sections above and below SC-35, additional 24-2 signals are observed (not shown), consistent with the  conclusions from the localization data presented in A–F. Bars, 3.5 μm (8 μm in the vertical dimension [C′ and D′]).
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Figure 5: Distribution of protein 4.1 epitopes in the interphase nucleus of human fibroblasts visualized by immunofluorescence. Location of the nucleus was confirmed in all cases by DAPI staining (not shown). (A and B) Double-label confocal microscopy of cells stained by protein 4.1 antibody 24-2 (green) and (A) antibody against nuclear pores (red) or (B) lamin B (red). The majority of nuclear protein 4.1 foci are interior to the periphery of the nucleus as demarcated by immunofluorescence of pores within nuclear membrane and by its subadjacent network of lamin B fibers. (Additional lamin B sites in the nuclear interior have been reported [Bridger et al., 1993; Moir et al., 1994]). Similar results were obtained by imaging epitopes for protein 4.1 antibodies 24-3, 10-1, and N-2 relative to antipore and antilamin epitopes. (C) Three-dimensional image analysis of nuclear epitopes probed with anti-RBC 80-kD 4.1 showing a 0.3-μm horizontal optical midsection of an immunofluorescent cell with a line indicating the position of the vertical reconstruction depicted in C′. Since optical resolution in the z-axis (vertical) is less than in the x-y (horizontal) axis, the reconstructed image is less sharp; the dome shape of the top of the cell and the flat bottom plane of the coverslip are somewhat obscured by the coincidence of out of focus light. (A cartoon depicting the approximate shape of the vertical cross-section through the nucleus is presented in the bottom panel.) Thus, it is apparent that epitopes for protein 4.1 lie in numerous planes within the volume of the cell. Similar results were obtained in three-dimensional reconstructions using the 24-2, 24-3, and 10-1 antibodies as probes. (D and D′) Double-label confocal microscopy of a fibroblast using anti–24-2 (green) and anti-NuMA (red). In a horizontal plane through the midsection of the cell (D), protein 4.1 foci appeared within the NuMA-stained nuclear interior. (D′) Three-dimensional reconstructions were made of three different vertical planes from this NuMA:24-2 double-labeled cell. The flattened plane of the coverslip appears at the top of the vertical reconstructed images. The conclusion that 4.1 epitopes localized at multiple planes throughout the volume of the nucleus is entirely consistent with the observations obtained in images A, B, and C. The same conclusion was reached when cells were probed with antibodies against NuMA and RBC 80-kD protein 4.1. (E and F) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against PCNA (red). Optical sections through the fibroblast nucleus show that 24-2 (green) and PCNA (red) epitopes coincided (yellow) in many areas except in the vicinities of the nucleoli, which either are relatively devoid of any signal or sometimes contained a clustering of 24-2 (green) signals. Both PCNA and 24-2 epitopes resided in multiple planes throughout the nucleus (not shown). (G and H) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against SC-35 (red). Optical sections are in the plane of the cell containing SC-35 domains; most SC-35 domains in fibroblasts are located in a single plane (Carter et al., 1993). It is apparent that the vast majority of the SC-35 domains contained areas of coincident staining with 24-2 (seen as yellow coloration), most often at the periphery of the SC-35 domains. In optical sections above and below SC-35, additional 24-2 signals are observed (not shown), consistent with the conclusions from the localization data presented in A–F. Bars, 3.5 μm (8 μm in the vertical dimension [C′ and D′]).
Mentions: The nucleus is bounded by two concentric lipid bilayers perforated by nuclear pores and an underlying ring of lamin, a specialized intermediate filament. Nuclear-associated 4.1 immunofluorescent signals could have arisen from cytoplasmic epitopes overlying the nucleus, from nuclear membrane sites, or from interior nuclear locations. In fact, since 80-kD protein 4.1 in mature erythrocytes is a component of the plasma membrane skeleton (Fig. 4), one might predict that 4.1 would localize to the nuclear membrane or even the underlying lamin protein ring. To distinguish among these possibilities, double-label confocal microscopy was performed to localize 4.1 foci relative to epitopes for two well-characterized nuclear proteins, lamin B and the nuclear pore complex. Optical horizontal sections through the center of the nucleus revealed well-separated rhodamine (Fig. 5 A, stain for lamin; Fig. 5 B, stain for pores) and FITC (stain for 4.1) signals, leading to the conclusion that 4.1 epitopes are within the area bounded by nuclear pores embedded in the nuclear membrane and by the underlying lamin protein ring. Furthermore, the smooth circumferential staining of both pores and lamin in the preparations rules out the possibility that internal 4.1 foci are being generated by invaginations of the nuclear membrane.

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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