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Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells.

Lechardeur D, Xu M, Lukacs GL - J. Cell Biol. (2004)

Bottom Line: We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD.The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells.Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells.

View Article: PubMed Central - PubMed

Affiliation: Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.

ABSTRACT
Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3-dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.

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Association of CAD with the nuclear matrix in apoptotic HeLa cells. (A) Biochemical isolation of nuclear matrix from cells expressing the CAD-GFP/ICAD-myc complex. Apoptosis was induced with STS for 2 h. HeLa cells were biochemically fractionated and the CAD-GFP content of each fraction was visualized by anti-GFP immunoblotting. Wcl, whole-cell lysate (lane 1); CSK, extraction with the cytoskeletal buffer (lane 2); DNaseI digestion (lane 3); NaCl, high salt extraction (lane 4); and nuclear matrix proteins (lane 5). (B and C) In situ isolation of the nuclear matrix was performed as in A on HeLa cells grown on glass coverslips. CAD-HA and chromatin was visualized by indirect immunostaining and with DAPI, respectively. Single x-y optical sections are shown. (D) Colocalization of CAD-GFP and CAD-HA with NuMA. Apoptosis was induced with STS for 2 h. HeLa cells were immunostained with anti-HA and anti-NuMa antibodies. (E) Colocalization of CAD-HA with NuMA. Immunostaining was performed as in D. Inset shows a magnification of the images captured with a fluorescence microscope (DMI RE2; Leica), deconvolved, and analyzed using OpenLab software (Improvision). Single images were selected for presentation. Bars, 10 μm.
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fig7: Association of CAD with the nuclear matrix in apoptotic HeLa cells. (A) Biochemical isolation of nuclear matrix from cells expressing the CAD-GFP/ICAD-myc complex. Apoptosis was induced with STS for 2 h. HeLa cells were biochemically fractionated and the CAD-GFP content of each fraction was visualized by anti-GFP immunoblotting. Wcl, whole-cell lysate (lane 1); CSK, extraction with the cytoskeletal buffer (lane 2); DNaseI digestion (lane 3); NaCl, high salt extraction (lane 4); and nuclear matrix proteins (lane 5). (B and C) In situ isolation of the nuclear matrix was performed as in A on HeLa cells grown on glass coverslips. CAD-HA and chromatin was visualized by indirect immunostaining and with DAPI, respectively. Single x-y optical sections are shown. (D) Colocalization of CAD-GFP and CAD-HA with NuMA. Apoptosis was induced with STS for 2 h. HeLa cells were immunostained with anti-HA and anti-NuMa antibodies. (E) Colocalization of CAD-HA with NuMA. Immunostaining was performed as in D. Inset shows a magnification of the images captured with a fluorescence microscope (DMI RE2; Leica), deconvolved, and analyzed using OpenLab software (Improvision). Single images were selected for presentation. Bars, 10 μm.

Mentions: Finally, the hypothesis that CAD binds to the chromatin-depleted subnuclear compartment, also called interchromatin space or nuclear matrix, was tested. Nuclear matrix preparation was obtained by the combination of successive detergent extraction, DNase digestion, and high salt washes of HeLa cells as described previously (Nickerson et al., 1997). Although CAD was largely depleted from the nuclear matrix preparation of nonapoptotic cells, a significant fraction of CAD-GFP remained associated with nonchromatin proteins of apoptotic cells according to immunoblot analysis (Fig. 7 A). The association of HA-CAD as well as CAD-GFP with nuclear interchromatin structures could be confirmed at the morphological level by fluorescence microscopy in apoptotic cells (Fig. 7, B and C), but not in control cells. CAD remained nuclear upon the complete digestion of the chromosomal DNA by DNase, verified by immunostaining of DAPI-negative nuclei (Fig. 7, B and C). In contrast, nuclear matrix preparations failed to retain GFP-NLS, visualized either by fluorescence microscopy or by immunoblotting (unpublished data). These observations are consistent with the notion that CAD, but not the heterodimeric CAD/ICAD, preferentially binds to the nuclear matrix of apoptotic cells.


Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells.

Lechardeur D, Xu M, Lukacs GL - J. Cell Biol. (2004)

Association of CAD with the nuclear matrix in apoptotic HeLa cells. (A) Biochemical isolation of nuclear matrix from cells expressing the CAD-GFP/ICAD-myc complex. Apoptosis was induced with STS for 2 h. HeLa cells were biochemically fractionated and the CAD-GFP content of each fraction was visualized by anti-GFP immunoblotting. Wcl, whole-cell lysate (lane 1); CSK, extraction with the cytoskeletal buffer (lane 2); DNaseI digestion (lane 3); NaCl, high salt extraction (lane 4); and nuclear matrix proteins (lane 5). (B and C) In situ isolation of the nuclear matrix was performed as in A on HeLa cells grown on glass coverslips. CAD-HA and chromatin was visualized by indirect immunostaining and with DAPI, respectively. Single x-y optical sections are shown. (D) Colocalization of CAD-GFP and CAD-HA with NuMA. Apoptosis was induced with STS for 2 h. HeLa cells were immunostained with anti-HA and anti-NuMa antibodies. (E) Colocalization of CAD-HA with NuMA. Immunostaining was performed as in D. Inset shows a magnification of the images captured with a fluorescence microscope (DMI RE2; Leica), deconvolved, and analyzed using OpenLab software (Improvision). Single images were selected for presentation. Bars, 10 μm.
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fig7: Association of CAD with the nuclear matrix in apoptotic HeLa cells. (A) Biochemical isolation of nuclear matrix from cells expressing the CAD-GFP/ICAD-myc complex. Apoptosis was induced with STS for 2 h. HeLa cells were biochemically fractionated and the CAD-GFP content of each fraction was visualized by anti-GFP immunoblotting. Wcl, whole-cell lysate (lane 1); CSK, extraction with the cytoskeletal buffer (lane 2); DNaseI digestion (lane 3); NaCl, high salt extraction (lane 4); and nuclear matrix proteins (lane 5). (B and C) In situ isolation of the nuclear matrix was performed as in A on HeLa cells grown on glass coverslips. CAD-HA and chromatin was visualized by indirect immunostaining and with DAPI, respectively. Single x-y optical sections are shown. (D) Colocalization of CAD-GFP and CAD-HA with NuMA. Apoptosis was induced with STS for 2 h. HeLa cells were immunostained with anti-HA and anti-NuMa antibodies. (E) Colocalization of CAD-HA with NuMA. Immunostaining was performed as in D. Inset shows a magnification of the images captured with a fluorescence microscope (DMI RE2; Leica), deconvolved, and analyzed using OpenLab software (Improvision). Single images were selected for presentation. Bars, 10 μm.
Mentions: Finally, the hypothesis that CAD binds to the chromatin-depleted subnuclear compartment, also called interchromatin space or nuclear matrix, was tested. Nuclear matrix preparation was obtained by the combination of successive detergent extraction, DNase digestion, and high salt washes of HeLa cells as described previously (Nickerson et al., 1997). Although CAD was largely depleted from the nuclear matrix preparation of nonapoptotic cells, a significant fraction of CAD-GFP remained associated with nonchromatin proteins of apoptotic cells according to immunoblot analysis (Fig. 7 A). The association of HA-CAD as well as CAD-GFP with nuclear interchromatin structures could be confirmed at the morphological level by fluorescence microscopy in apoptotic cells (Fig. 7, B and C), but not in control cells. CAD remained nuclear upon the complete digestion of the chromosomal DNA by DNase, verified by immunostaining of DAPI-negative nuclei (Fig. 7, B and C). In contrast, nuclear matrix preparations failed to retain GFP-NLS, visualized either by fluorescence microscopy or by immunoblotting (unpublished data). These observations are consistent with the notion that CAD, but not the heterodimeric CAD/ICAD, preferentially binds to the nuclear matrix of apoptotic cells.

Bottom Line: We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD.The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells.Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells.

View Article: PubMed Central - PubMed

Affiliation: Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.

ABSTRACT
Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3-dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.

Show MeSH
Related in: MedlinePlus