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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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The immobilization of CAD in the apoptotic nucleus is caspase-3 dependent. (A) Effect of caspase inhibitors on CAD-GFP diffusional mobility. HeLa cells were exposed to STS for 3 h in the presence of DEVD-CHO (20 μM) or ZVAD-fmk (50 μM). Immobile fraction was determined by FRAP as described in Fig. 5. (B and C) CAD-GFP is immobilized only in exogenous caspase-3–expressing MCF7 cells. (B) x-y optical sections of MCF7 cells, cotransfected with CAD-GFP, ICAD-myc, and procaspase-3. Cells were incubated for 3.5 h with STS before the caspase-3 substrate Red-DEVD-fmk was added to the medium to identify cells containing only CAD-GFP (−casp3) or both CAD-GFP and active caspase-3 (+casp3). CAD-GFP mobility was measured by FLIP as described in Fig. 3 D. Bar, 10 μM. (C) Results from B were plotted as in Fig. 3 D. Control represents a nontreated MCF7 cell.
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fig6: The immobilization of CAD in the apoptotic nucleus is caspase-3 dependent. (A) Effect of caspase inhibitors on CAD-GFP diffusional mobility. HeLa cells were exposed to STS for 3 h in the presence of DEVD-CHO (20 μM) or ZVAD-fmk (50 μM). Immobile fraction was determined by FRAP as described in Fig. 5. (B and C) CAD-GFP is immobilized only in exogenous caspase-3–expressing MCF7 cells. (B) x-y optical sections of MCF7 cells, cotransfected with CAD-GFP, ICAD-myc, and procaspase-3. Cells were incubated for 3.5 h with STS before the caspase-3 substrate Red-DEVD-fmk was added to the medium to identify cells containing only CAD-GFP (−casp3) or both CAD-GFP and active caspase-3 (+casp3). CAD-GFP mobility was measured by FLIP as described in Fig. 3 D. Bar, 10 μM. (C) Results from B were plotted as in Fig. 3 D. Control represents a nontreated MCF7 cell.

Mentions: Several lines of evidence suggested that the immobilization of CAD required caspase-3 activation: (1) treatment of the cells with the pan-caspase inhibitor Z-VAD-fmk, or with the caspase-3–specific inhibitor DEVD-CHO abolished the immobilization of CAD upon STS treatment (Fig. 6 A); (2) the diffusional mobility of CAD was preserved in STS-treated MCF7 cells that lack functional caspase-3 (Fig. 6 B, top; Fig. 6 C and Video 1), but CAD-GFP was immobilized in MCF7 cells expressing functional caspase-3 (Fig. 6 B, bottom; Fig. 6 C); (3) immobilization of activated CAD is not restricted to cells undergoing STS-induced apoptosis: CAD was also immobilized to comparable extent in HeLa cells subjected to UV irradiation or CHX and TNFα treatment (Fig. S3, B, C, F, and G; available at http://www.jcb.org/cgi/content/full/jcb.200404105/DC1). Both inducers provoked the degradation of ICAD (Fig. S3, A and E) and their effects on CAD immobilization were prevented in the presence of the caspase-3 inhibitor DEVD-CHO (Fig. S3, D and H), implying that CAD activation occurred via the caspase-3–dependent pathway. Collectively, these results suggest that immobilization of CAD-GFP is a common feature of nuclei undergoing caspase-3–dependent apoptosis regardless of the upstream signaling mechanism.


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

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

The immobilization of CAD in the apoptotic nucleus is caspase-3 dependent. (A) Effect of caspase inhibitors on CAD-GFP diffusional mobility. HeLa cells were exposed to STS for 3 h in the presence of DEVD-CHO (20 μM) or ZVAD-fmk (50 μM). Immobile fraction was determined by FRAP as described in Fig. 5. (B and C) CAD-GFP is immobilized only in exogenous caspase-3–expressing MCF7 cells. (B) x-y optical sections of MCF7 cells, cotransfected with CAD-GFP, ICAD-myc, and procaspase-3. Cells were incubated for 3.5 h with STS before the caspase-3 substrate Red-DEVD-fmk was added to the medium to identify cells containing only CAD-GFP (−casp3) or both CAD-GFP and active caspase-3 (+casp3). CAD-GFP mobility was measured by FLIP as described in Fig. 3 D. Bar, 10 μM. (C) Results from B were plotted as in Fig. 3 D. Control represents a nontreated MCF7 cell.
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fig6: The immobilization of CAD in the apoptotic nucleus is caspase-3 dependent. (A) Effect of caspase inhibitors on CAD-GFP diffusional mobility. HeLa cells were exposed to STS for 3 h in the presence of DEVD-CHO (20 μM) or ZVAD-fmk (50 μM). Immobile fraction was determined by FRAP as described in Fig. 5. (B and C) CAD-GFP is immobilized only in exogenous caspase-3–expressing MCF7 cells. (B) x-y optical sections of MCF7 cells, cotransfected with CAD-GFP, ICAD-myc, and procaspase-3. Cells were incubated for 3.5 h with STS before the caspase-3 substrate Red-DEVD-fmk was added to the medium to identify cells containing only CAD-GFP (−casp3) or both CAD-GFP and active caspase-3 (+casp3). CAD-GFP mobility was measured by FLIP as described in Fig. 3 D. Bar, 10 μM. (C) Results from B were plotted as in Fig. 3 D. Control represents a nontreated MCF7 cell.
Mentions: Several lines of evidence suggested that the immobilization of CAD required caspase-3 activation: (1) treatment of the cells with the pan-caspase inhibitor Z-VAD-fmk, or with the caspase-3–specific inhibitor DEVD-CHO abolished the immobilization of CAD upon STS treatment (Fig. 6 A); (2) the diffusional mobility of CAD was preserved in STS-treated MCF7 cells that lack functional caspase-3 (Fig. 6 B, top; Fig. 6 C and Video 1), but CAD-GFP was immobilized in MCF7 cells expressing functional caspase-3 (Fig. 6 B, bottom; Fig. 6 C); (3) immobilization of activated CAD is not restricted to cells undergoing STS-induced apoptosis: CAD was also immobilized to comparable extent in HeLa cells subjected to UV irradiation or CHX and TNFα treatment (Fig. S3, B, C, F, and G; available at http://www.jcb.org/cgi/content/full/jcb.200404105/DC1). Both inducers provoked the degradation of ICAD (Fig. S3, A and E) and their effects on CAD immobilization were prevented in the presence of the caspase-3 inhibitor DEVD-CHO (Fig. S3, D and H), implying that CAD activation occurred via the caspase-3–dependent pathway. Collectively, these results suggest that immobilization of CAD-GFP is a common feature of nuclei undergoing caspase-3–dependent apoptosis regardless of the upstream signaling mechanism.

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