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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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Immobilization of CAD in apoptotic nucleus. The mobility of CAD-GFP was monitored by spot photobleaching in the nucleus of transiently transfected HeLa cells exposed to STS for the indicated time. (A and B) FRAP of a single nucleus at different times of apoptosis. (A) Single x-y optical sections of nucleus. The circles indicate the bleached area. Bar (A), 5 μm. The corresponding recovery curves are plotted in B. Note that after 2 h of apoptosis, recovery in the bleached areas is negligible (indicated by arrowheads). (C and D) Phosphatidylserine translocation and CAD-GFP mobility. FRAP was performed as in A on cells incubated with Alexa 594–labeled annexin V. Cells were visualized by differential interference contrast microscopy. The corresponding recovery curves are shown in D. Bar (C), 10 μm.
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fig4: Immobilization of CAD in apoptotic nucleus. The mobility of CAD-GFP was monitored by spot photobleaching in the nucleus of transiently transfected HeLa cells exposed to STS for the indicated time. (A and B) FRAP of a single nucleus at different times of apoptosis. (A) Single x-y optical sections of nucleus. The circles indicate the bleached area. Bar (A), 5 μm. The corresponding recovery curves are plotted in B. Note that after 2 h of apoptosis, recovery in the bleached areas is negligible (indicated by arrowheads). (C and D) Phosphatidylserine translocation and CAD-GFP mobility. FRAP was performed as in A on cells incubated with Alexa 594–labeled annexin V. Cells were visualized by differential interference contrast microscopy. The corresponding recovery curves are shown in D. Bar (C), 10 μm.

Mentions: The nuclear retention of both HA-CAD and CAD-GFP in permeabilized apoptotic cells is consistent with the notion that the diffusion of CAD became restricted during apoptosis (Fig. 1 A and Fig. 2 E). To further characterize this process, FRAP measurements were performed by spot photobleaching on HeLa nuclei. After an initial lag period (1–1.5 h), the immobile fraction of CAD-GFP progressively increased in the nucleus of a single cell (Fig. 4, A and B). To verify that CAD immobilization requires the activation of the apoptotic enzyme cascade, the translocation of phosphatidylserine into the outer leaflet of the plasma membrane was measured simultaneously with CAD mobility on STS-treated cells. Remarkably, the immobilization of CAD was observed only in those cells that displayed annexin V staining and plasma membrane blebbing (Fig. 4, C and D). To confirm this notion, the mobility of CAD was determined on a large number cells after induction of apoptosis, as well as in cells deficient of functional caspase-3.


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

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

Immobilization of CAD in apoptotic nucleus. The mobility of CAD-GFP was monitored by spot photobleaching in the nucleus of transiently transfected HeLa cells exposed to STS for the indicated time. (A and B) FRAP of a single nucleus at different times of apoptosis. (A) Single x-y optical sections of nucleus. The circles indicate the bleached area. Bar (A), 5 μm. The corresponding recovery curves are plotted in B. Note that after 2 h of apoptosis, recovery in the bleached areas is negligible (indicated by arrowheads). (C and D) Phosphatidylserine translocation and CAD-GFP mobility. FRAP was performed as in A on cells incubated with Alexa 594–labeled annexin V. Cells were visualized by differential interference contrast microscopy. The corresponding recovery curves are shown in D. Bar (C), 10 μm.
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Related In: Results  -  Collection

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

fig4: Immobilization of CAD in apoptotic nucleus. The mobility of CAD-GFP was monitored by spot photobleaching in the nucleus of transiently transfected HeLa cells exposed to STS for the indicated time. (A and B) FRAP of a single nucleus at different times of apoptosis. (A) Single x-y optical sections of nucleus. The circles indicate the bleached area. Bar (A), 5 μm. The corresponding recovery curves are plotted in B. Note that after 2 h of apoptosis, recovery in the bleached areas is negligible (indicated by arrowheads). (C and D) Phosphatidylserine translocation and CAD-GFP mobility. FRAP was performed as in A on cells incubated with Alexa 594–labeled annexin V. Cells were visualized by differential interference contrast microscopy. The corresponding recovery curves are shown in D. Bar (C), 10 μm.
Mentions: The nuclear retention of both HA-CAD and CAD-GFP in permeabilized apoptotic cells is consistent with the notion that the diffusion of CAD became restricted during apoptosis (Fig. 1 A and Fig. 2 E). To further characterize this process, FRAP measurements were performed by spot photobleaching on HeLa nuclei. After an initial lag period (1–1.5 h), the immobile fraction of CAD-GFP progressively increased in the nucleus of a single cell (Fig. 4, A and B). To verify that CAD immobilization requires the activation of the apoptotic enzyme cascade, the translocation of phosphatidylserine into the outer leaflet of the plasma membrane was measured simultaneously with CAD mobility on STS-treated cells. Remarkably, the immobilization of CAD was observed only in those cells that displayed annexin V staining and plasma membrane blebbing (Fig. 4, C and D). To confirm this notion, the mobility of CAD was determined on a large number cells after induction of apoptosis, as well as in cells deficient of functional caspase-3.

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