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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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Extractability of CAD from normal and apoptotic HeLa cells. (A–C) Indirect immunofluorescence of (A) HA-CAD/ICAD-myc, (B) GFP-NLS, and (C) histones in HeLa cells. The indicated proteins were immunostained in cells fixed with PFA before (−perm) or after (+perm) permeabilization in cytoskeletal buffer containing 0.2% Triton X-100. DNA was stained with DAPI. Apoptosis was induced with STS for 2 h. Single optical sections were acquired with a fluorescence microscope (DMI RE2; Leica) and OpenLab software. Bars, 10 μm. (D) Nuclear retention of endogenous CAD. Lysates were prepared from normal and apoptotic HeLa cells before (lane 1) or 5 min after (lane 3) permeabilization as in A–C. Extracellular release of CAD from the cells is shown in lanes 2 and 4. Apoptosis was induced with STS for 2 h. Cell lysates and medium corresponding to the same number of cells was processed for Western blot using an anti-CAD antibody.
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fig1: Extractability of CAD from normal and apoptotic HeLa cells. (A–C) Indirect immunofluorescence of (A) HA-CAD/ICAD-myc, (B) GFP-NLS, and (C) histones in HeLa cells. The indicated proteins were immunostained in cells fixed with PFA before (−perm) or after (+perm) permeabilization in cytoskeletal buffer containing 0.2% Triton X-100. DNA was stained with DAPI. Apoptosis was induced with STS for 2 h. Single optical sections were acquired with a fluorescence microscope (DMI RE2; Leica) and OpenLab software. Bars, 10 μm. (D) Nuclear retention of endogenous CAD. Lysates were prepared from normal and apoptotic HeLa cells before (lane 1) or 5 min after (lane 3) permeabilization as in A–C. Extracellular release of CAD from the cells is shown in lanes 2 and 4. Apoptosis was induced with STS for 2 h. Cell lysates and medium corresponding to the same number of cells was processed for Western blot using an anti-CAD antibody.

Mentions: We used epitope-tagged CAD/ICAD to investigate whether the CAD/ICAD heterodimer binds to nuclear constituents by immunostaining. Both HA-tagged CAD and ICAD-myc transiently expressed in HeLa cells. Both proteins were confined to the nuclei (Fig. 1 A, left, −perm; Lechardeur et al., 2000). Permeabilization of the cells by the nonionic detergent Triton X-100 before fixation provoked the complete loss of HA-CAD/ICAD-myc from the nuclei of normal cells (Fig. 1 A, left, +perm). EGFP harboring three tandem repeats of the NLS of the SV40-antigen (GFP-NLS) was also depleted, validating the permeabilization procedure (Fig. 1 B, left; +perm). In contrast, association of core and linker histones with the chromosomal DNA was retained in permeabilized cells (Fig. 1 C, left; +perm). Collectively, these observations suggest the CAD/ICAD complex can be released from the nuclei similarly to soluble proteins such as GFP-NLS.


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

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

Extractability of CAD from normal and apoptotic HeLa cells. (A–C) Indirect immunofluorescence of (A) HA-CAD/ICAD-myc, (B) GFP-NLS, and (C) histones in HeLa cells. The indicated proteins were immunostained in cells fixed with PFA before (−perm) or after (+perm) permeabilization in cytoskeletal buffer containing 0.2% Triton X-100. DNA was stained with DAPI. Apoptosis was induced with STS for 2 h. Single optical sections were acquired with a fluorescence microscope (DMI RE2; Leica) and OpenLab software. Bars, 10 μm. (D) Nuclear retention of endogenous CAD. Lysates were prepared from normal and apoptotic HeLa cells before (lane 1) or 5 min after (lane 3) permeabilization as in A–C. Extracellular release of CAD from the cells is shown in lanes 2 and 4. Apoptosis was induced with STS for 2 h. Cell lysates and medium corresponding to the same number of cells was processed for Western blot using an anti-CAD antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172457&req=5

fig1: Extractability of CAD from normal and apoptotic HeLa cells. (A–C) Indirect immunofluorescence of (A) HA-CAD/ICAD-myc, (B) GFP-NLS, and (C) histones in HeLa cells. The indicated proteins were immunostained in cells fixed with PFA before (−perm) or after (+perm) permeabilization in cytoskeletal buffer containing 0.2% Triton X-100. DNA was stained with DAPI. Apoptosis was induced with STS for 2 h. Single optical sections were acquired with a fluorescence microscope (DMI RE2; Leica) and OpenLab software. Bars, 10 μm. (D) Nuclear retention of endogenous CAD. Lysates were prepared from normal and apoptotic HeLa cells before (lane 1) or 5 min after (lane 3) permeabilization as in A–C. Extracellular release of CAD from the cells is shown in lanes 2 and 4. Apoptosis was induced with STS for 2 h. Cell lysates and medium corresponding to the same number of cells was processed for Western blot using an anti-CAD antibody.
Mentions: We used epitope-tagged CAD/ICAD to investigate whether the CAD/ICAD heterodimer binds to nuclear constituents by immunostaining. Both HA-tagged CAD and ICAD-myc transiently expressed in HeLa cells. Both proteins were confined to the nuclei (Fig. 1 A, left, −perm; Lechardeur et al., 2000). Permeabilization of the cells by the nonionic detergent Triton X-100 before fixation provoked the complete loss of HA-CAD/ICAD-myc from the nuclei of normal cells (Fig. 1 A, left, +perm). EGFP harboring three tandem repeats of the NLS of the SV40-antigen (GFP-NLS) was also depleted, validating the permeabilization procedure (Fig. 1 B, left; +perm). In contrast, association of core and linker histones with the chromosomal DNA was retained in permeabilized cells (Fig. 1 C, left; +perm). Collectively, these observations suggest the CAD/ICAD complex can be released from the nuclei similarly to soluble proteins such as GFP-NLS.

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