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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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Interfering with nuclear entrapment of CAD facilitates its extracellular release from apoptotic cells. (A) HeLa cells, transiently expressing the mouse HA-CAD/ICAD-myc or the HA-CADΔNLS/ICADΔNLS-myc complex were exposed to STS for 2.5 h in serum-free medium. CAD was immunoprecipitated from both the medium (med) and the cell lysate (cell) with anti-HA antibody. The precipitates and the cell lysates (lys) were probed with anti-CAD and anti-myc antibodies to visualize exogenous CAD and ICAD. (B) As a control, HeLa cells were transfected with the HA-CADΔNLS/ICADΔNLS-myc and were incubated overnight in serum-free medium. CAD was then immunoprecipitated from the medium and probed with the indicated antibodies.
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fig9: Interfering with nuclear entrapment of CAD facilitates its extracellular release from apoptotic cells. (A) HeLa cells, transiently expressing the mouse HA-CAD/ICAD-myc or the HA-CADΔNLS/ICADΔNLS-myc complex were exposed to STS for 2.5 h in serum-free medium. CAD was immunoprecipitated from both the medium (med) and the cell lysate (cell) with anti-HA antibody. The precipitates and the cell lysates (lys) were probed with anti-CAD and anti-myc antibodies to visualize exogenous CAD and ICAD. (B) As a control, HeLa cells were transfected with the HA-CADΔNLS/ICADΔNLS-myc and were incubated overnight in serum-free medium. CAD was then immunoprecipitated from the medium and probed with the indicated antibodies.

Mentions: To prevent the immobilization of CAD, we took advantage of our previous observation demonstrating that deletion of the NLS in both CAD and ICAD caused the relocation of the CAD-ΔNLS/ICAD-ΔNLS complex into the cytoplasm and prevented nuclear uptake of CAD-ΔNLS (Lechardeur et al., 2000). Importantly, neither the dimerization nor the activation kinetics of CAD was altered by deleting the two NLSs (unpublished data). The extracellular liberation of HA-CAD from apoptotic HeLa cells expressing either CAD/ICAD or CAD-ΔNLS/ICAD-ΔNLS was determined by immunoblotting after the immunoprecipitation of the extracellular CAD with anti-HA antibody (Fig. 9). A significant fraction of CAD-ΔNLS, but not CAD, leaked into the medium from HeLa cells undergoing STS-induced apoptosis (Fig. 9, lanes 3 and 6). The absence of myc-tagged ICAD in the immunoprecipitates confirmed that the proteolysis of ICAD-myc preceded the extracellular egress of CAD (Fig. 9, bottom). Thus, nuclear entrapment of CAD may have a critical role in preventing the extracellular release of the active DNase from apoptotic cells.


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

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

Interfering with nuclear entrapment of CAD facilitates its extracellular release from apoptotic cells. (A) HeLa cells, transiently expressing the mouse HA-CAD/ICAD-myc or the HA-CADΔNLS/ICADΔNLS-myc complex were exposed to STS for 2.5 h in serum-free medium. CAD was immunoprecipitated from both the medium (med) and the cell lysate (cell) with anti-HA antibody. The precipitates and the cell lysates (lys) were probed with anti-CAD and anti-myc antibodies to visualize exogenous CAD and ICAD. (B) As a control, HeLa cells were transfected with the HA-CADΔNLS/ICADΔNLS-myc and were incubated overnight in serum-free medium. CAD was then immunoprecipitated from the medium and probed with the indicated antibodies.
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Related In: Results  -  Collection

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fig9: Interfering with nuclear entrapment of CAD facilitates its extracellular release from apoptotic cells. (A) HeLa cells, transiently expressing the mouse HA-CAD/ICAD-myc or the HA-CADΔNLS/ICADΔNLS-myc complex were exposed to STS for 2.5 h in serum-free medium. CAD was immunoprecipitated from both the medium (med) and the cell lysate (cell) with anti-HA antibody. The precipitates and the cell lysates (lys) were probed with anti-CAD and anti-myc antibodies to visualize exogenous CAD and ICAD. (B) As a control, HeLa cells were transfected with the HA-CADΔNLS/ICADΔNLS-myc and were incubated overnight in serum-free medium. CAD was then immunoprecipitated from the medium and probed with the indicated antibodies.
Mentions: To prevent the immobilization of CAD, we took advantage of our previous observation demonstrating that deletion of the NLS in both CAD and ICAD caused the relocation of the CAD-ΔNLS/ICAD-ΔNLS complex into the cytoplasm and prevented nuclear uptake of CAD-ΔNLS (Lechardeur et al., 2000). Importantly, neither the dimerization nor the activation kinetics of CAD was altered by deleting the two NLSs (unpublished data). The extracellular liberation of HA-CAD from apoptotic HeLa cells expressing either CAD/ICAD or CAD-ΔNLS/ICAD-ΔNLS was determined by immunoblotting after the immunoprecipitation of the extracellular CAD with anti-HA antibody (Fig. 9). A significant fraction of CAD-ΔNLS, but not CAD, leaked into the medium from HeLa cells undergoing STS-induced apoptosis (Fig. 9, lanes 3 and 6). The absence of myc-tagged ICAD in the immunoprecipitates confirmed that the proteolysis of ICAD-myc preceded the extracellular egress of CAD (Fig. 9, bottom). Thus, nuclear entrapment of CAD may have a critical role in preventing the extracellular release of the active DNase from apoptotic cells.

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