Characterization of mitochondrial cell death pathways in聽C. elegans

The identification of CPS-6/EndoG, an apoptotic nuclease from mitochondria

Mitochondrial endonuclease G is important for apoptosis in聽颁.听别濒别驳补苍蝉

JAY聽PARRISH*, LILY聽LI#, KRISTINA聽KLOTZ*, DUNCAN聽LEDWICH*, XIAODONG聽WANG#聽& DING聽XUE*

*聽Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA

聽Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into聽internucleosomal聽repeats. Here we report the characterization of the聽cps-6聽gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of聽颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉聽in the DNA degradation process during apoptosis. cps-6聽encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in聽颁.听别濒别驳补苍蝉. Reduction of聽cps-6聽activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in聽颁.听别濒别驳补苍蝉. This observation provides聽in vivo聽evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to聽be involved in programmed cell death in聽颁.听别濒别驳补苍蝉, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

Parrish, J., Li, L., Klotz, K.,聽Ledwich, D., Wang, X.D., and Xue, D. (2001).听聽颁.听别濒别驳补苍蝉聽mitochondrial endonuclease G is important for apoptosis.听聽狈补迟耻谤别听412, 90-94.听(听补苍诲听笔顿贵).

WAH-1, a聽颁.听别濒别驳补苍蝉聽AIF聽homologue,聽cooperates with CPS-6/EndoG聽to regulates apoptotic DNA degradation.

Mechanisms of AIF-mediated apoptotic DNA degradation in聽颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉

Xiaochen聽Wang,1聽Chonglin聽Yang,1聽Jijie聽Chai,2聽Yigong聽Shi,2聽Ding Xue1

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA
  2. Department聽of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

Apoptosis-inducing factor (AIF), a mitochondrial聽oxidoreductase, is released into the cytoplasm to induce cell death in response to apoptotic signals. However, the mechanisms underlying this process have not been resolved. We report that inactivation of the聽颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉聽AIF homolog wah-1 by RNA interference delayed the normal progression of apoptosis and caused a defect in apoptotic DNA degradation. WAH-1 localized in聽颁.听别濒别驳补苍蝉聽mitochondria and was released into the cytosol and nucleus by the BH3-domain protein EGL-1 in a聽caspase听(CED-3)-dependent manner.听聽In addition, WAH-1 associated and cooperated with the mitochondrial endonuclease CPS-6/endonuclease G (EndoG) to promote DNA degradation and apoptosis. Thus, AIF and聽EndoG聽define a single, mitochondria-initiated apoptotic DNA degradation pathway that is conserved between聽颁.听别濒别驳补苍蝉聽and mammals.

Wang, X.C., Yang, C.L., Cai, J.J., Shi, Y.G., and Xue, D. (2002).听Mechanisms of AIF-mediated apoptotic DNA degradation in聽Caenorhabditis elegans.听Science聽298, 1587-1592.听(听补苍诲听PDF).听听(PDF)听

WAH-1 also promotes聽phosphatidylserine聽externalization in apoptotic cells through SCRM-1

颁.听别濒别驳补苍蝉聽mitochondrial factor WAH-1 promotes聽phosphatidylserine聽externalization in apoptotic cells through phospholipid聽scramblase聽SCRM-1

Xiaochen聽Wang1, Jin Wang2, Keiko Gengyo-Ando3,聽Lichuan聽Gu4, Chun-Ling Sun1,聽Chonglin聽Yang1, Yong Shi1, Tetsuo Kobayashi3,聽Yigong聽Shi4,聽Shohei聽Mitani3, Xiao-Song Xie2聽&聽Ding Xue1

  1. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
  2. McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  3. Department of Physiology, Tokyo Women's Medical University, School of Medicine, and CREST, JST, Tokyo, 162-8666, Japan.
  4. Department聽of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

Externalization of聽phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the聽phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an聽annexin聽V-based聽phosphatidylserine聽labelling聽method and show that a majority of apoptotic germ cells in聽颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉聽have surface-exposed聽phosphatidylserine, indicating that聽phosphatidylserine聽externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the聽颁.听别濒别驳补苍蝉聽apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial聽apoptogenic聽factor, or the聽颁.听别濒别驳补苍蝉聽phospholipid聽scramblase聽1 (SCRM-1), a plasma membrane protein, reduces聽phosphatidylserine聽exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity聽in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane聽phosphatidylserine聽externalization through its downstream effector, SCRM-1.

Wang, X.C., Wang, J., Gengyo-Ando, K., Gu, L.C., Sun, C.L., Yang, C.L., Shi, Y., Kobayashi, T., Shi, Y.G., Mitani, S., Xie, X.S., and Xue, D. (2007). "颁.听别濒别驳补苍蝉聽mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1".听Nature Cell Biology聽9, 541-549.听(听补苍诲听PDF).听听(PDF)

Identification of two additional mitochondrial pathways leading to elimination of mitochondria during apoptosis

颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉聽drp-1听补苍诲听fis-2聽Regulate Distinct Cell-Death Execution Pathways Downstream of聽ced-3聽and Independent of聽ced-9

David G. Breckenridge1,聽Byung-Ho Kang1, 2, David Kokel1,聽Shohei聽Mitani3, L. Andrew Staehelin1聽and Ding Xue1

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA
  2. Department of Microbiology and Cell Science, Integrated Center for Biotechnology Research, University of Florida, Gainesville, FL 32608, USA
  3. Department of Physiology, Tokyo Women's Medical University, School of Medicine, and CREST, JST, Tokyo, 162-8666, Japan

The聽dynamin聽family of聽GTPases聽regulate聽mitochondrial fission and fusion processes and have been implicated in controlling the release of聽caspase聽activators from mitochondria during apoptosis. Here we report that profusion genes聽fzo-1听补苍诲听eat-3聽or the聽profission聽gene聽drp-1聽are聽not required for apoptosis activation in聽颁.听别濒别驳补苍蝉. However, minor聽proapoptotic聽roles for聽drp-1听补苍诲听fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds.听drp-1听补苍诲听fis-2聽function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3聽caspase聽to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1's聽proapoptotic聽function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in聽颁.听别濒别驳补苍蝉聽and reveal distinct roles for聽drp-1听补苍诲听fis-2聽as mediators of cell-death execution downstream of聽caspase聽activation.

Breckenridge, D., Kang, B.H., Kokel, D., Mitani, S., Staehelin, A.L., and Xue, D. (2008).听Caenorhabditis elegans聽drp-1听补苍诲听fis-2聽regulate distinct cell death execution pathways downstream of聽ced-3聽and independent of聽ced-9.听Mol. Cell聽31 586-597.听(听补苍诲听PDF)听Developmental Cell Preview (PDF)

The roles of聽颁.听别濒别驳补苍蝉聽Bcl-2 proteins EGL-1 and CED-9 in regulating mitochondrial dynamics

Bcl-2 proteins EGL-1 and CED-9 do not regulate mitochondrial fission or fusion in聽颁补别苍辞谤丑补产诲颈迟颈蝉听别濒别驳补苍蝉

David G. Breckenridge1*聽#,聽Byung-Ho Kang2聽#, and Ding Xue1*

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA
  2. Department of Microbiology and Cell Science, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32611, USA

The Bcl-2 family proteins are critical apoptosis regulators that associate with mitochondria and control the activation of聽caspases.听聽Recently, both mammalian and聽颁.听别濒别驳补苍蝉聽Bcl-2 proteins have been implicated in controlling mitochondrial fusion and fission processes in both living and apoptotic cells.听聽To better understand the potential roles of Bcl-2 family proteins in regulating mitochondrial dynamics, we carried out a detailed analysis of mitochondria in animals that either lose or have increased activity of聽egl-1听补苍诲听ced-9, two Bcl-2 family genes that induce and inhibit apoptosis in聽颁.听别濒别驳补苍蝉,聽respectively.听聽Unexpectedly, we found that loss of聽egl-1聽or聽ced-9, or overexpression of their gene products, had no apparent effect on mitochondrial connectivity or mitochondrial size. Moreover, loss of聽ced-9聽did not affect the mitochondrial morphology observed in a聽drp-1聽mutant, where mitochondrial fusion occurs but mitochondrial fission is defective, or in a聽fzo-1聽mutant, where mitochondrial fission occurs but mitochondrial fusion is restricted, suggesting that聽ced-9聽is not required for either the mitochondrial fission or fusion process in聽颁.听别濒别驳补苍蝉.听聽Taken together, our results argue against an evolutionarily conserved role for Bcl-2 proteins in regulating mitochondrial fission and fusion.

Breckenridge, D., Kang, B.H., and Xue, D. (2009). Bcl-2 proteins EGL-1 and CED-9 do not regulate mitochondrial fission or fusion in聽Caenorhabditis elegans.听Current Biology聽19, 768-773.听(听补苍诲听PDF)

For detailed review on mitochondrial cell death pathways and studies:

Breckenridge, D. and Xue, D. (2004).听Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases.听Curr. Opin. Cell Biol.聽16, 647-652. (听补苍诲听PDF)