Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death

Science

Volumn 287, Issue 5457, 2000, Pages 1485-1489

  Chen, Fangli ^a   Hersh, Bradley M ^a   Conradt, Barbara ^a   Zhou, Zheng ^a   Riemer, Dieter ^a   Gruenbaum, Yosef ^a   Horvitz, H Robert ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CASPASE; CELL PROTEIN; TRANSCRIPTION FACTOR;

APOPTOSIS; ARTICLE; CAENORHABDITIS ELEGANS; CELL NUCLEUS MEMBRANE; CELLULAR DISTRIBUTION; EMBRYO; GENE MUTATION; INTRACELLULAR TRANSPORT; NONHUMAN; PRIORITY JOURNAL; PROTEIN TRANSPORT;

AMINO ACID SUBSTITUTION; ANIMALS; ANIMALS, GENETICALLY MODIFIED; APOPTOSIS; APOPTOSIS REGULATORY PROTEINS; CAENORHABDITIS ELEGANS; CAENORHABDITIS ELEGANS PROTEINS; CALCIUM-BINDING PROTEINS; CASPASES; CYSTEINE ENDOPEPTIDASES; GENES, HELMINTH; HELMINTH PROTEINS; IMMUNOHISTOCHEMISTRY; MITOCHONDRIA; MUTATION; NUCLEAR ENVELOPE; PHENOTYPE; PROTO-ONCOGENE PROTEINS; REPRESSOR PROTEINS;

CAENORHABDITIS ELEGANS; INVERTEBRATA;

EID: 0034712042     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5457.1485     Document Type: Article

References (42)

1. M. D. Jacobson, M. Weil, M. C. Raff, Cell 88, 347 (1997).
2. C. B. Thompson, Science 267, 1456 (1995).
3. M. M. Metzstein, C. M. Stanfield, H. R. Horvitz, Trends Genet. 14, 410 (1998).
4. M. O. Hengartner and H. R. Horvitz, Cell 76, 665 (1994).
5. B. Conradt and H. R. Horvitz, Cell 93, 519 (1998).
6. J. Yuan et al., Cell 75, 641 (1993).
7. H. Zou et al., Cell 90, 405 (1997).
8. D Wu, H. D. Wallen, C. Nuñez, Science 275, 1126 (1997); M. Irmler et al., FEBS Lett. 406, 189 (1997); A. M. Chinnaiyan et al., Science 275, 1122 (1997); M. S. Spector et al., Nature 385, 653 (1997).
9. D Wu, H. D. Wallen, C. Nuñez, Science 275, 1126 (1997); M. Irmler et al., FEBS Lett. 406, 189 (1997); A. M. Chinnaiyan et al., Science 275, 1122 (1997); M. S. Spector et al., Nature 385, 653 (1997).
10. D Wu, H. D. Wallen, C. Nuñez, Science 275, 1126 (1997); M. Irmler et al., FEBS Lett. 406, 189 (1997); A. M. Chinnaiyan et al., Science 275, 1122 (1997); M. S. Spector et al., Nature 385, 653 (1997).
11. D Wu, H. D. Wallen, C. Nuñez, Science 275, 1126 (1997); M. Irmler et al., FEBS Lett. 406, 189 (1997); A. M. Chinnaiyan et al., Science 275, 1122 (1997); M. S. Spector et al., Nature 385, 653 (1997).
12. L. del Peso, V. M. Gonzalez, G. Nunez, J. Biol. Chem. 273, 33495 (1998).
13. S. Seshagiri and L K. Miller, Curr. Biol. 7, 455 (1997).
14. 2+-nitrilotriacetic acid-agarose (Qiagen) and injected with RIBI adjuvant into rabbits. The antiserum was affinity-purified against His-10-CED-9 immobilized on nitrocellulose. For the generation of anti-CED-4, full-length ced-4S cDNA was doned into pXHA [S. J. Elledge et al., Proc. Natl. Acad. Sci. U.S.A. 89, 2907 (1992)]. The hemaggtutinin-CED-4 fusion was purified from indusion bodies of Escherichia coli and injected into rabbits and rats. The rabbit antiserum was used without further purification, and rat antiserum was affinity-purified against glutathione-S- transferase-CED-4 immobilized on nitrocellulose. Embyros were collected by bleaching mixed-stage worms in a 0.8 N NaOH, 8% hypochlorite solution. Embryos were fixed and permeabilized essentially as described [C. Guenther and G. Garriga, Development 122, 3509 (1996)]. Embryos were incubated in a 1:100 dilution of primary antibody, washed four times, incubated for 2 hours in a 1:25 dilution of secondary antibody, washed four times, washed once in phosphate-buffered saline plus 4′,6′-diamidino-2-phenylindole (DAPI, 1 μg/ml), and resuspended in an equal volume of VECTASHIELD mounting medium (Vector Labs). Embryos for mitochondrial colocalization experiments were collected from worms grown in the dark on NGM agar plates containing MitoTracker Red CMXRos (2 μg/ml, Molecular Probes).
15. 2+-nitrilotriacetic acid-agarose (Qiagen) and injected with RIBI adjuvant into rabbits. The antiserum was affinity-purified against His- 10-CED-9 immobilized on nitrocellulose. For the generation of anti-CED-4, full-length ced-4S cDNA was doned into pXHA [S. J. Elledge et al., Proc. Natl. Acad. Sci. U.S.A. 89, 2907 (1992)]. The hemaggtutinin-CED-4 fusion was purified from indusion bodies of Escherichia coli and injected into rabbits and rats. The rabbit antiserum was used without further purification, and rat antiserum was affinity-purified against glutathione-S-transferase-CED-4 immobilized on nitrocellulose. Embyros were collected by bleaching mixed-stage worms in a 0.8 N NaOH, 8% hypochlorite solution. Embryos were fixed and permeabilized essentially as described [C. Guenther and G. Garriga, Development 122, 3509 (1996)]. Embryos were incubated in a 1:100 dilution of primary antibody, washed four times, incubated for 2 hours in a 1:25 dilution of secondary antibody, washed four times, washed once in phosphate-buffered saline plus 4′,6′-diamidino-2-phenylindole (DAPI, 1 μg/ml), and resuspended in an equal volume of VECTASHIELD mounting medium (Vector Labs). Embryos for mitochondrial colocalization experiments were collected from worms grown in the dark on NGM agar plates containing MitoTracker Red CMXRos (2 μg/ml, Molecular Probes).
16. Supplementary data can be found in Web figure 1 at www.sciencemag.org/feature/data/1046764.shl.
17. Mutant strains carrying the following alleles of cell-death genes were used in this study; ced-1(e1735.), engulfment-defective; ced-3(n717), splice acceptor mutation, exon 7; ced-9(n2812), Q46amber; ced-9(n1950), G169E; ced-9(n1950 n2161), ced-9(n1950 n2077), loss-of-function mutations; ced-9(n1653), Y149N; ced-4(n1162), Q79ochre; ced-4(n2860), E263K; ced-4(n2879), E276K; ced-4(n3040), P23L; ced-4(n3043), D20N; ced-4(n3100). S339P; ced-4(n3141), R53K; egt-1(n1084), G-to-A nucleotide transition at nucleotide +5631; egl-1(n1084 n3082), n 1084 lesion plus 5-base pair (bp) deletion in egl-1 coding region.
18. M. Poot et al., J. Histochem. Cytochem. 44, 1363 (1996).
19. J. Yuan and H. R. Horvitz, Development 116, 309 (1992).
20. D. Riemer, H. Dodemont, K. Weber, Eur. J. Cell Biol. 62, 214 (1993).
21. Supplementary data can be found in Web figure 2 at www.sciencemag.org/feature/data/1046764.shl.
22. Embryos for fractionation were collected as described (11). Embryos were resuspended in five volumes of cold hypotonic buffer (10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethyisulfonyl fluoride, 250 mM sucrose) and homogenized in a 1-ml Dounce tissue grinder. The homogenates were centrifuged at 40g briefly to remove worm debris. The supernatant was centrifuged twice at 750g for 10 min, and the resulting pellets were pooled as the nuclear fraction. The supernatant was further centrifuged at 100,000g for 1 hour. The pellet was designated the organelle and membrane fraction and the supernatant the soluble cytosolic S100 fraction. The pooled nuclear fraction was washed once with homogenization buffer. One-fifth of each fraction was used for immunoblotting analysis. Rabbit polyclonal antibody directed against human acetylated histone H4 (Upstate Biotechnology) was used as a marker for the nuclear fraction. Monoclonal anti-Ce HSP90 was used as a marker for the cytosolic fraction.
23. D. D. Newmeyer, D. M. Farschon, J. C. Reed, Cell 79, 353 (1994).
24. hsp egl-1 (S) was injected at a concentration of 2 ng/μl, along with p76-16B [L. Bloom and H. R. Horvitz, Proc. Natl. Acad. Sci. U.S.A. 94, 3414 (1997)] at 50 ng/μl, into a ced-1(e1735); egl-1(n1084 n3082) unc-76(e911) strain as described [C. Mello and A. Fire, Methods Cell Biol. 46, 451 (1995)]. For immunofluorescence, mixed-stage transgenic worms were incubated at 33°C for 1 hour followed by a 2-hour recovery at 20°C Embryos were then collected as in (77). For analysis of cell corpses, transgenic adults were allowed to lay eggs at 20°C for 2 hours, subjected to a 1-hour heat shock at 33°C, allowed to lay eggs for an additional 2 hours at 20°C, and then removed from the plates. Hatched L1 transgenic animals were examined by Nomarski microscopy for corpses in the head.
25. hsp egl-1 (S) was injected at a concentration of 2 ng/μl, along with p76-16B [L. Bloom and H. R. Horvitz, Proc. Natl. Acad. Sci. U.S.A. 94, 3414 (1997)] at 50 ng/μl, into a ced-1(e1735); egl-1(n1084 n3082) unc-76(e911) strain as described [C. Mello and A. Fire, Methods Cell Biol. 46, 451 (1995)]. For immunofluorescence, mixed-stage transgenic worms were incubated at 33°C for 1 hour followed by a 2-hour recovery at 20°C Embryos were then collected as in (77). For analysis of cell corpses, transgenic adults were allowed to lay eggs at 20°C for 2 hours, subjected to a 1-hour heat shock at 33°C, allowed to lay eggs for an additional 2 hours at 20°C, and then removed from the plates. Hatched L1 transgenic animals were examined by Nomarski microscopy for corpses in the head.
26. E. M. Hedgecock, J. E. Sulston, J. N. Thomson, Science 220, 1277 (1983).
27. F. Chen, B. M. Hersh, H. R. Horvitz, data not shown.
28. B. M. Hersh, B. Conradt, H. R. Horvitz, data not shown.
29. S. Ottilie et al., Cell Death Differ. 4, 526 (1997).
30. M. Nguyen et al., J. Biol. Chem. 268, 25265 (1993); Y. Akao et al., Cancer Res. 54, 2468 (1994); S. Krajewski et al., Cancer Res. 53, 4701 (1993).
31. M. Nguyen et al., J. Biol. Chem. 268, 25265 (1993); Y. Akao et al., Cancer Res. 54, 2468 (1994); S. Krajewski et al., Cancer Res. 53, 4701 (1993).
32. M. Nguyen et al., J. Biol. Chem. 268, 25265 (1993); Y. Akao et al., Cancer Res. 54, 2468 (1994); S. Krajewski et al., Cancer Res. 53, 4701 (1993).
33. C. Pan, K. O'Rourke, V. M. Dixit, J. Biol. Chem. 273, 5841 (1998); Y. Hu et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4386 (1998).
34. C. Pan, K. O'Rourke, V. M. Dixit, J. Biol. Chem. 273, 5841 (1998); Y. Hu et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4386 (1998).
35. K. Moriishi et al., Proc. Natl. Acad. Sci. U.S.A. 96, 9683 (1999).
36. B. Conradt and H. R. Horvitz, Cell 98, 317 (1999).
37. D. Xue and H. R. Horvitz, Nature 390, 305 (1997).
38. K. G. Wolter et al., J. Cell Biol. 139, 1281 (1997).
39. S. A. Susin et al., J. Exp. Med. 189, 381 (1999); M. Mancini et al., J. Cell Biol. 140, 1485 (1998); B. Zhivotovsky, A. Samali, A. Gahm, S. Orrenius, Cell Death Differ. 6, 644 (1999).
40. S. A. Susin et al., J. Exp. Med. 189, 381 (1999); M. Mancini et al., J. Cell Biol. 140, 1485 (1998); B. Zhivotovsky, A. Samali, A. Gahm, S. Orrenius, Cell Death Differ. 6, 644 (1999).
41. S. A. Susin et al., J. Exp. Med. 189, 381 (1999); M. Mancini et al., J. Cell Biol. 140, 1485 (1998); B. Zhivotovsky, A. Samali, A. Gahm, S. Orrenius, Cell Death Differ. 6, 644 (1999).
42. We thank members of the Horvitz laboratory for helpful comments and discussions concerning this manuscript. We thank Y. Yamaguchi and J. Miwa for anti-Ce HSP90 monoclonal antibody; D. Xue for generating pET19b-CED-9ΔC; B. Davies, A. Madi, S. Shaham, E. Speliotes, and C. Stanfield for ced-4 missense mutations; B. Castor for DNA sequence determinations; and M. Boxern and S. van den Heuvel for assistance with microscopy. B.M.H. was supported by a Howard Hughes Predoctoral Fellowship. B.C. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and a Leukemia Society Special Fellowship. Z.Z. was supported by a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (DRC 1343). H.R.H. is an Investigator of the Howard Hughes Medical Institute.