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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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Localization of the nuclear matrix targeting motif in CAD. (A) Schematic pictures of deletion mutants of CAD fused to the NH2 terminus of EGFP. All constructs were freely mobile in nonapoptotic cells. The relative mobilities of the truncated CAD in STS-treated cells are indicated by the size of the immobile pool on the right, representing at least 10 independent measurements for each construct. No or negligible effect, −; partial immobilization, +; and complete immobilization, ++. (B) Mutant CAD-GFP fusion proteins were transiently expressed in HeLa cells. The apparent molecular mass of fusion proteins was assessed by immunoblotting using an anti-GFP antibody. (C) FRAP studies of selected CAD-GFP fusion proteins. The mobility of the indicated fusion proteins was determined with FRAP as described in Fig. 3, before and after STS (2 h) treatment. Representative categories of the CAD chimera in apoptotic cells are demonstrated. CIDE-GFP, marginal inhibition of mobility (left); CAD[84–152]-GFP, partial immobilization (middle); and CAD[1–152], complete immobilization (right).
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fig8: Localization of the nuclear matrix targeting motif in CAD. (A) Schematic pictures of deletion mutants of CAD fused to the NH2 terminus of EGFP. All constructs were freely mobile in nonapoptotic cells. The relative mobilities of the truncated CAD in STS-treated cells are indicated by the size of the immobile pool on the right, representing at least 10 independent measurements for each construct. No or negligible effect, −; partial immobilization, +; and complete immobilization, ++. (B) Mutant CAD-GFP fusion proteins were transiently expressed in HeLa cells. The apparent molecular mass of fusion proteins was assessed by immunoblotting using an anti-GFP antibody. (C) FRAP studies of selected CAD-GFP fusion proteins. The mobility of the indicated fusion proteins was determined with FRAP as described in Fig. 3, before and after STS (2 h) treatment. Representative categories of the CAD chimera in apoptotic cells are demonstrated. CIDE-GFP, marginal inhibition of mobility (left); CAD[84–152]-GFP, partial immobilization (middle); and CAD[1–152], complete immobilization (right).

Mentions: To elucidate the signal responsible for CAD association with the nuclear matrix, progressive COOH- and NH2-terminal truncations of CAD were generated and fused in frame to EGFP (Fig. 8 A), and the mobility of the chimeras was measured. Truncated variants of human CAD were designed based on sequence alignments of the human, mouse, chicken, and Drosophila CAD, respecting putative domain boundaries (Mukae et al., 2000) (Fig. 8 A). Western blot analysis verified the expected molecular mass of the truncated CAD-GFP fusion proteins (Fig. 8 B). The nuclear mobility of truncated CAD was assayed in proliferating and apoptotic HeLa cells. Four of the nine fusion proteins that were immobilized in STS-treated HeLa cells contained amino acid residues between 84 and 152 of CAD (CAD[1–152], [1–278], [84–152], and [84–203]) (Fig. 8, A and C). In contrast, fusion proteins incorporating peptide fragments of the COOH terminus of CAD ([152–203], [203–278], [278–338], and [203–338]) or the CIDE-N domain remained as mobile as GFP-NLS in apoptotic cells (Fig. 8 A and unpublished data). The partial immobilization of CAD[84–152] and CAD[84–203] was potentiated in the presence of the CIDE-N domain because both CAD[1–152] and CAD[1–278] were immobile in apoptotic nuclei (Fig. 8 C). These results suggest that the NH2-terminal 152 amino acid residues are necessary for the immobilization of CAD.


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

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

Localization of the nuclear matrix targeting motif in CAD. (A) Schematic pictures of deletion mutants of CAD fused to the NH2 terminus of EGFP. All constructs were freely mobile in nonapoptotic cells. The relative mobilities of the truncated CAD in STS-treated cells are indicated by the size of the immobile pool on the right, representing at least 10 independent measurements for each construct. No or negligible effect, −; partial immobilization, +; and complete immobilization, ++. (B) Mutant CAD-GFP fusion proteins were transiently expressed in HeLa cells. The apparent molecular mass of fusion proteins was assessed by immunoblotting using an anti-GFP antibody. (C) FRAP studies of selected CAD-GFP fusion proteins. The mobility of the indicated fusion proteins was determined with FRAP as described in Fig. 3, before and after STS (2 h) treatment. Representative categories of the CAD chimera in apoptotic cells are demonstrated. CIDE-GFP, marginal inhibition of mobility (left); CAD[84–152]-GFP, partial immobilization (middle); and CAD[1–152], complete immobilization (right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172457&req=5

fig8: Localization of the nuclear matrix targeting motif in CAD. (A) Schematic pictures of deletion mutants of CAD fused to the NH2 terminus of EGFP. All constructs were freely mobile in nonapoptotic cells. The relative mobilities of the truncated CAD in STS-treated cells are indicated by the size of the immobile pool on the right, representing at least 10 independent measurements for each construct. No or negligible effect, −; partial immobilization, +; and complete immobilization, ++. (B) Mutant CAD-GFP fusion proteins were transiently expressed in HeLa cells. The apparent molecular mass of fusion proteins was assessed by immunoblotting using an anti-GFP antibody. (C) FRAP studies of selected CAD-GFP fusion proteins. The mobility of the indicated fusion proteins was determined with FRAP as described in Fig. 3, before and after STS (2 h) treatment. Representative categories of the CAD chimera in apoptotic cells are demonstrated. CIDE-GFP, marginal inhibition of mobility (left); CAD[84–152]-GFP, partial immobilization (middle); and CAD[1–152], complete immobilization (right).
Mentions: To elucidate the signal responsible for CAD association with the nuclear matrix, progressive COOH- and NH2-terminal truncations of CAD were generated and fused in frame to EGFP (Fig. 8 A), and the mobility of the chimeras was measured. Truncated variants of human CAD were designed based on sequence alignments of the human, mouse, chicken, and Drosophila CAD, respecting putative domain boundaries (Mukae et al., 2000) (Fig. 8 A). Western blot analysis verified the expected molecular mass of the truncated CAD-GFP fusion proteins (Fig. 8 B). The nuclear mobility of truncated CAD was assayed in proliferating and apoptotic HeLa cells. Four of the nine fusion proteins that were immobilized in STS-treated HeLa cells contained amino acid residues between 84 and 152 of CAD (CAD[1–152], [1–278], [84–152], and [84–203]) (Fig. 8, A and C). In contrast, fusion proteins incorporating peptide fragments of the COOH terminus of CAD ([152–203], [203–278], [278–338], and [203–338]) or the CIDE-N domain remained as mobile as GFP-NLS in apoptotic cells (Fig. 8 A and unpublished data). The partial immobilization of CAD[84–152] and CAD[84–203] was potentiated in the presence of the CIDE-N domain because both CAD[1–152] and CAD[1–278] were immobile in apoptotic nuclei (Fig. 8 C). These results suggest that the NH2-terminal 152 amino acid residues are necessary for the immobilization of CAD.

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