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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 diffusional mobility of CAD-GFP and GFP-NLS in HeLa cells undergoing apoptosis. FRAP experiments and confocal imaging were conducted as described in Fig. 3. (A) Qualitative FRAP during apoptosis. Representative nuclei of different cells, expressing CAD-GFP, were imaged before and during the recovery after photobleaching the boxed area. Near-complete immobilization of CAD-GFP was observed after 3 h of STS treatment. Bar, 5 μm. (B) Half-time of fluorescence recoveries and (C) immobile fractions of CAD-GFP and GFP-NLS after STS exposure for the indicated duration. Half-time of recoveries were calculated from normalized recovery curves of 30–40 individual cells for each time interval. The mobile fraction was calculated as the percentage of corrected total fluorescence intensity reached during the recovery phase (see Materials and methods).
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fig5: The diffusional mobility of CAD-GFP and GFP-NLS in HeLa cells undergoing apoptosis. FRAP experiments and confocal imaging were conducted as described in Fig. 3. (A) Qualitative FRAP during apoptosis. Representative nuclei of different cells, expressing CAD-GFP, were imaged before and during the recovery after photobleaching the boxed area. Near-complete immobilization of CAD-GFP was observed after 3 h of STS treatment. Bar, 5 μm. (B) Half-time of fluorescence recoveries and (C) immobile fractions of CAD-GFP and GFP-NLS after STS exposure for the indicated duration. Half-time of recoveries were calculated from normalized recovery curves of 30–40 individual cells for each time interval. The mobile fraction was calculated as the percentage of corrected total fluorescence intensity reached during the recovery phase (see Materials and methods).

Mentions: As illustrated by the time-lapse images of CAD-GFP recoveries (Fig. 5 A) and the statistical analysis, STS provoked a modest immobilization of CAD during the first 2 h of apoptosis (Fig. 5, B and C). Extending the incubation time to 3 h increased the t1/2 by >20 fold (t1/2 = 23 ± 4.6 s; n = 18) relative to the control (t1/2 = 1.0 ± 0.05 s; n = 39) (Fig. 5 B). A fraction of CAD (40 ± 4.2%; n = 20) became immobile between 2–3 h of apoptosis induction (Fig. 5 C). Extending the incubation up to 3 h immobilized >80% of CAD-GFP (Fig. 5 C). Similar nuclear immobilization was observed in BHK and MEF ICAD−/− cells, as well as using the mouse CAD-GFP fusion protein, suggesting that CAD immobilization is cell- and species-independent (unpublished data). Inhibition of CAD diffusion was not restricted to specific regions, but affected the entire nucleus, as demonstrated by FLIP analysis (Video 1 and Fig. S2, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200404105/DC1). In contrast, the diffusional mobility of GFP-NLS was not altered in apoptotic cells, as shown by the rapid fluorescence recovery rate and insignificant immobile fraction of GFP-NLS after 3 h of STS treatment (Fig. 5, B and C; Fig. S2). The lack of GFP-NLS immobilization cannot be attributed to the slower onset of nuclear apoptosis because a negligible effect on the mobility of GFP-NLS was observed in cells coexpressing HA-CAD/ICAD or using the DNase-deficient CADH242N-GFP fusion protein (unpublished data and see following paragraph).


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 diffusional mobility of CAD-GFP and GFP-NLS in HeLa cells undergoing apoptosis. FRAP experiments and confocal imaging were conducted as described in Fig. 3. (A) Qualitative FRAP during apoptosis. Representative nuclei of different cells, expressing CAD-GFP, were imaged before and during the recovery after photobleaching the boxed area. Near-complete immobilization of CAD-GFP was observed after 3 h of STS treatment. Bar, 5 μm. (B) Half-time of fluorescence recoveries and (C) immobile fractions of CAD-GFP and GFP-NLS after STS exposure for the indicated duration. Half-time of recoveries were calculated from normalized recovery curves of 30–40 individual cells for each time interval. The mobile fraction was calculated as the percentage of corrected total fluorescence intensity reached during the recovery phase (see Materials and methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172457&req=5

fig5: The diffusional mobility of CAD-GFP and GFP-NLS in HeLa cells undergoing apoptosis. FRAP experiments and confocal imaging were conducted as described in Fig. 3. (A) Qualitative FRAP during apoptosis. Representative nuclei of different cells, expressing CAD-GFP, were imaged before and during the recovery after photobleaching the boxed area. Near-complete immobilization of CAD-GFP was observed after 3 h of STS treatment. Bar, 5 μm. (B) Half-time of fluorescence recoveries and (C) immobile fractions of CAD-GFP and GFP-NLS after STS exposure for the indicated duration. Half-time of recoveries were calculated from normalized recovery curves of 30–40 individual cells for each time interval. The mobile fraction was calculated as the percentage of corrected total fluorescence intensity reached during the recovery phase (see Materials and methods).
Mentions: As illustrated by the time-lapse images of CAD-GFP recoveries (Fig. 5 A) and the statistical analysis, STS provoked a modest immobilization of CAD during the first 2 h of apoptosis (Fig. 5, B and C). Extending the incubation time to 3 h increased the t1/2 by >20 fold (t1/2 = 23 ± 4.6 s; n = 18) relative to the control (t1/2 = 1.0 ± 0.05 s; n = 39) (Fig. 5 B). A fraction of CAD (40 ± 4.2%; n = 20) became immobile between 2–3 h of apoptosis induction (Fig. 5 C). Extending the incubation up to 3 h immobilized >80% of CAD-GFP (Fig. 5 C). Similar nuclear immobilization was observed in BHK and MEF ICAD−/− cells, as well as using the mouse CAD-GFP fusion protein, suggesting that CAD immobilization is cell- and species-independent (unpublished data). Inhibition of CAD diffusion was not restricted to specific regions, but affected the entire nucleus, as demonstrated by FLIP analysis (Video 1 and Fig. S2, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200404105/DC1). In contrast, the diffusional mobility of GFP-NLS was not altered in apoptotic cells, as shown by the rapid fluorescence recovery rate and insignificant immobile fraction of GFP-NLS after 3 h of STS treatment (Fig. 5, B and C; Fig. S2). The lack of GFP-NLS immobilization cannot be attributed to the slower onset of nuclear apoptosis because a negligible effect on the mobility of GFP-NLS was observed in cells coexpressing HA-CAD/ICAD or using the DNase-deficient CADH242N-GFP fusion protein (unpublished data and see following paragraph).

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