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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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Nuclear dynamics of CAD in dividing HeLa cells. (A) Photobleaching of nuclear CAD-GFP, GFP-NLS, GFP, and H1.1-GFP in transiently transfected HeLa and MEF ICAD−/− cells. FRAP was performed as described in Materials and methods. Images show single x-y sections of the nucleus and were obtained before (pre) and after the bleach at indicated times. Although the laser intensity was sufficient to cause complete loss of fluorescence in PFA fixed cells, partial recovery occurred during the first scan due to the rapid lateral diffusion of the GFP chimeras. (B) Quantitative FRAP. Normalized fluorescence recovery was plotted as a function of time. Negligible recovery occurs on PFA-fixed HeLa cells (+PFA). (C) Diffusional coefficients of GFP and GFP chimeras in the nucleus of HeLa cells. The recovery curves were fitted according to a one-dimensional diffusion formula described in Materials and methods. Data are means ± SEM; n is indicated in brackets. (D) FLIP of CAD-GFP and GFP in HeLa cells. A 2-μm circle in the nucleus of HeLa cells expressing CAD-GFP or GFP was bleached in every 10 s. The entire nucleus was imaged after each bleach. Scans of the remaining fluorescence in the whole nucleus are shown for CAD-GFP– and GFP-expressing cells for the indicated times after the first bleach. Bar, 5 μm. (E) Quantitative FLIP. The ratio of the average fluorescence intensity of the half nucleus opposite to the bleach spot relative to the prebleach image was plotted at each time point. (F) Diffusional coefficients of CAD-GFP/ICAD in the nucleus of HeLa, BHK, MCF7, and MEF ICAD−/− cells. Diffusional coefficients were calculated as described in C. Data are means ± SEM.
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fig3: Nuclear dynamics of CAD in dividing HeLa cells. (A) Photobleaching of nuclear CAD-GFP, GFP-NLS, GFP, and H1.1-GFP in transiently transfected HeLa and MEF ICAD−/− cells. FRAP was performed as described in Materials and methods. Images show single x-y sections of the nucleus and were obtained before (pre) and after the bleach at indicated times. Although the laser intensity was sufficient to cause complete loss of fluorescence in PFA fixed cells, partial recovery occurred during the first scan due to the rapid lateral diffusion of the GFP chimeras. (B) Quantitative FRAP. Normalized fluorescence recovery was plotted as a function of time. Negligible recovery occurs on PFA-fixed HeLa cells (+PFA). (C) Diffusional coefficients of GFP and GFP chimeras in the nucleus of HeLa cells. The recovery curves were fitted according to a one-dimensional diffusion formula described in Materials and methods. Data are means ± SEM; n is indicated in brackets. (D) FLIP of CAD-GFP and GFP in HeLa cells. A 2-μm circle in the nucleus of HeLa cells expressing CAD-GFP or GFP was bleached in every 10 s. The entire nucleus was imaged after each bleach. Scans of the remaining fluorescence in the whole nucleus are shown for CAD-GFP– and GFP-expressing cells for the indicated times after the first bleach. Bar, 5 μm. (E) Quantitative FLIP. The ratio of the average fluorescence intensity of the half nucleus opposite to the bleach spot relative to the prebleach image was plotted at each time point. (F) Diffusional coefficients of CAD-GFP/ICAD in the nucleus of HeLa, BHK, MCF7, and MEF ICAD−/− cells. Diffusional coefficients were calculated as described in C. Data are means ± SEM.

Mentions: To compare the dynamics of CAD-GFP/ICAD with freely moving GFP and GFP-NLS in live cells, we measured their one-dimensional diffusion coefficient and mobile fraction by FRAP technique in HeLa cells. More than 80% of the initial fluorescence of CAD-GFP as well as GFP-NLS was recovered in 2–3 s, whereas complete recovery of GFP was attained in <1 s after the bleach (Fig. 3, A and B). CAD-GFP diffusional coefficient (DCAD-GFP = 1.34 ± 0.20 μm2/s; n = 32, mean ± SEM) was comparable to that of GFP-NLS (DGFP-NLS = 0.94 ± 0.10 μm2/s; n = 47), but was 6.6-fold slower than GFP (DGFP = 8.95 ± 0.95 μm2/s; n = 36) (Fig. 3 C). In comparison, the diffusional coefficient of histone H1 (H1.1-GFP,DH1.1GFP = 0.019 ± 0.003 μm2/s; n = 20) was ∼70-fold slower (Fig. 3, A and C). The slow mobility of H1.1-GFP confirms previously published results (Misteli et al., 2000; Kimura and Cook, 2001).


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

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

Nuclear dynamics of CAD in dividing HeLa cells. (A) Photobleaching of nuclear CAD-GFP, GFP-NLS, GFP, and H1.1-GFP in transiently transfected HeLa and MEF ICAD−/− cells. FRAP was performed as described in Materials and methods. Images show single x-y sections of the nucleus and were obtained before (pre) and after the bleach at indicated times. Although the laser intensity was sufficient to cause complete loss of fluorescence in PFA fixed cells, partial recovery occurred during the first scan due to the rapid lateral diffusion of the GFP chimeras. (B) Quantitative FRAP. Normalized fluorescence recovery was plotted as a function of time. Negligible recovery occurs on PFA-fixed HeLa cells (+PFA). (C) Diffusional coefficients of GFP and GFP chimeras in the nucleus of HeLa cells. The recovery curves were fitted according to a one-dimensional diffusion formula described in Materials and methods. Data are means ± SEM; n is indicated in brackets. (D) FLIP of CAD-GFP and GFP in HeLa cells. A 2-μm circle in the nucleus of HeLa cells expressing CAD-GFP or GFP was bleached in every 10 s. The entire nucleus was imaged after each bleach. Scans of the remaining fluorescence in the whole nucleus are shown for CAD-GFP– and GFP-expressing cells for the indicated times after the first bleach. Bar, 5 μm. (E) Quantitative FLIP. The ratio of the average fluorescence intensity of the half nucleus opposite to the bleach spot relative to the prebleach image was plotted at each time point. (F) Diffusional coefficients of CAD-GFP/ICAD in the nucleus of HeLa, BHK, MCF7, and MEF ICAD−/− cells. Diffusional coefficients were calculated as described in C. Data are means ± SEM.
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fig3: Nuclear dynamics of CAD in dividing HeLa cells. (A) Photobleaching of nuclear CAD-GFP, GFP-NLS, GFP, and H1.1-GFP in transiently transfected HeLa and MEF ICAD−/− cells. FRAP was performed as described in Materials and methods. Images show single x-y sections of the nucleus and were obtained before (pre) and after the bleach at indicated times. Although the laser intensity was sufficient to cause complete loss of fluorescence in PFA fixed cells, partial recovery occurred during the first scan due to the rapid lateral diffusion of the GFP chimeras. (B) Quantitative FRAP. Normalized fluorescence recovery was plotted as a function of time. Negligible recovery occurs on PFA-fixed HeLa cells (+PFA). (C) Diffusional coefficients of GFP and GFP chimeras in the nucleus of HeLa cells. The recovery curves were fitted according to a one-dimensional diffusion formula described in Materials and methods. Data are means ± SEM; n is indicated in brackets. (D) FLIP of CAD-GFP and GFP in HeLa cells. A 2-μm circle in the nucleus of HeLa cells expressing CAD-GFP or GFP was bleached in every 10 s. The entire nucleus was imaged after each bleach. Scans of the remaining fluorescence in the whole nucleus are shown for CAD-GFP– and GFP-expressing cells for the indicated times after the first bleach. Bar, 5 μm. (E) Quantitative FLIP. The ratio of the average fluorescence intensity of the half nucleus opposite to the bleach spot relative to the prebleach image was plotted at each time point. (F) Diffusional coefficients of CAD-GFP/ICAD in the nucleus of HeLa, BHK, MCF7, and MEF ICAD−/− cells. Diffusional coefficients were calculated as described in C. Data are means ± SEM.
Mentions: To compare the dynamics of CAD-GFP/ICAD with freely moving GFP and GFP-NLS in live cells, we measured their one-dimensional diffusion coefficient and mobile fraction by FRAP technique in HeLa cells. More than 80% of the initial fluorescence of CAD-GFP as well as GFP-NLS was recovered in 2–3 s, whereas complete recovery of GFP was attained in <1 s after the bleach (Fig. 3, A and B). CAD-GFP diffusional coefficient (DCAD-GFP = 1.34 ± 0.20 μm2/s; n = 32, mean ± SEM) was comparable to that of GFP-NLS (DGFP-NLS = 0.94 ± 0.10 μm2/s; n = 47), but was 6.6-fold slower than GFP (DGFP = 8.95 ± 0.95 μm2/s; n = 36) (Fig. 3 C). In comparison, the diffusional coefficient of histone H1 (H1.1-GFP,DH1.1GFP = 0.019 ± 0.003 μm2/s; n = 20) was ∼70-fold slower (Fig. 3, A and C). The slow mobility of H1.1-GFP confirms previously published results (Misteli et al., 2000; Kimura and Cook, 2001).

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