Mitochondrial Cell Death Pathways

Characterization of mitochondrial cell death pathways in C. elegans

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

Mitochondrial endonuclease G is important for apoptosis in C. elegans

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

* Department of Molecular, Cellular, and Developmental Biology, University of Colorado, 麻豆影院, Colorado 80309, USA

 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 internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 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 C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X.D., and Xue, D. (2001).  C. elegans mitochondrial endonuclease G is important for apoptosis.  Nature 412, 90-94. ( and PDF).

WAH-1, a C. elegans AIF homologue, cooperates with CPS-6/EndoG to regulates apoptotic DNA degradation.

Mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans

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

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, 麻豆影院, CO 80309, USA
  2. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

Apoptosis-inducing factor (AIF), a mitochondrial 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 Caenorhabditis elegans 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 C. elegans mitochondria and was released into the cytosol and nucleus by the BH3-domain protein EGL-1 in a caspase (CED-3)-dependent manner.  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 EndoG define a single, mitochondria-initiated apoptotic DNA degradation pathway that is conserved between C. elegans and mammals.

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

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

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

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

  1. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, 麻豆影院, 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 of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

Externalization of 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 phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine 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 in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine 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). "C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1". Nature Cell Biology 9, 541-549. ( and ).  ()

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

Caenorhabditis elegans drp-1 and fis-2 Regulate Distinct Cell-Death Execution Pathways Downstream of ced-3 and Independent of ced-9

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

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, 麻豆影院, 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 dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase 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 proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

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

The roles of C. elegans 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 Caenorhabditis elegans

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

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, 麻豆影院, 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 caspases.  Recently, both mammalian and C. elegans Bcl-2 proteins have been implicated in controlling mitochondrial fusion and fission processes in both living and apoptotic cells.  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 egl-1 and ced-9, two Bcl-2 family genes that induce and inhibit apoptosis in C. elegans, respectively.  Unexpectedly, we found that loss of egl-1 or ced-9, or overexpression of their gene products, had no apparent effect on mitochondrial connectivity or mitochondrial size. Moreover, loss of ced-9 did not affect the mitochondrial morphology observed in a drp-1 mutant, where mitochondrial fusion occurs but mitochondrial fission is defective, or in a fzo-1 mutant, where mitochondrial fission occurs but mitochondrial fusion is restricted, suggesting that ced-9 is not required for either the mitochondrial fission or fusion process in C. elegans.  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 Caenorhabditis elegans. Current Biology 19, 768-773. ( and )

For detailed review on mitochondrial cell death pathways and studies:

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