1) Identification and characterization of downstream pathways of the CED-3 cell death protease 

Analysis of downstream pathways of important enzymatic biomolecules (e.g., kinases and proteases) that have multiple substrates has always been a difficult challenge. Targets of these enzymes could be difficult to identify through conventional genetic screens, since elimination of one of the multiple targets of an enzyme may fail to cause any visible phenotype that can be scored in genetic screens.  Enzymatic targets could also be difficult to identify through commonly used biochemical approaches such as protein interaction screens that require stable interaction rarely seen between an enzyme and a substrate.  In fact, to date there have been no good methods available that can systematically and effectively address the problem of substrate identification.

 

During apoptosis, a family of aspartate-specific cysteine proteases named caspases are activated proteolytically to execute the apoptotic program.  Little is known about the identities of their in vivo targets or their downstream pathways that mediate the killing functions.  We have developed a novel, GFP-based genetic screen to identify downstream targets of the C. elegans caspase, CED-3.  In this screen, the green fluorescent protein (GFP) and a constitutively activated version of CED-3 were co-expressed in C. elegans mechanosensory neurons and GFP was used as a sensitive Òcell existingÓ marker to isolate mutations that either partially suppress or delay ectopic neuronal deaths caused by the activated CED-3 (Figure 1).  These CED-3 protease suppressors (we named these cps mutations) likely affect genes that act downstream of, or in parallel to, ced-3 to mediate various cell-killing processes.  We have screened approximately 150,000 C. elegans haploid genomes and isolated more than 80 cps mutations.   Phenotypic analyses of these cps mutants suggest that these mutations not only suppress CED-3-induced ectopic neuronal deaths but also affect normal programmed cell death in C. elegans, indicating that they are true cell-death mutants.  Genetic analyses of these cps mutations indicate that they affect at least fourteen new cell death genes (cps-1 to cps-14) and one previously identified gene (ced-1), which acts downstream of ced-3 to remove apoptotic cell corpses.  Phenotypic analyses of these cps mutants suggest that they can be categorized into three major groups: 1) mutations that weakly suppress cell deaths in C. elegans (cps-1 and cps-2), 2) mutations that delay the normal progression of apoptosis (cps-3, 4, 5, 6, 7, 8, 10, and 11), and 3) mutations that may cause defect in the removal of cell corpses (cps-9, 12, 13, 14).  These 14 cps genes may encode important components that act downstream of the CED-3 protease to regulate or execute different aspects of the cell disassembly and removal process (Figure 2).  Indeed, using the TUNEL assay (an assay used to detect DNA breaks generated during apoptosis), we found that cps-3, 4, 6, 7, 8, 10, 11, 13 and 14 genes are involved in the chromosome fragmentation process, one of the key steps and a hallmark of apoptosis.  In addition, using an annexin V staining assay (used to detect surface-exposed phosphatidylserine), we found that three genes (cps-12, cps-13, and cps-14) affect externalization of phosphatidylserine (PS) in apoptotic cells.  PS normally is restricted to the inner leaflet of the plasma membrane but is exposed or flipped out in apoptotic cells and has been suggested to serve as an engulfment signal for phagocytosis.  How PS is flipped out in dying cells during apoptosis has not been clear and is a topic of intense studies.  Molecular characterization of cps-12, cps-13 and cps-14 will likely provide important insights towards addressing this fundamental biological question.

 

We recently cloned the cps-6 gene and found that cps-6 encodes a homologue of human mitochondrial endonuclease G (endoG).  In collaboration with Dr. Xiaodong WangÕs group at the University of Texas Southwestern Medical Center, we showed that the CPS-6 protein localizes to C. elegans mitochondria and possesses an endonuclease activity that is capable of inducing the generation of apoptotic DNA ladders in isolated Hela cell nuclei.  Furthermore, the mouse EndoG can substitute for the functions of cps-6 in C. elegans, suggesting that CPS-6 and EndoG define an evolutionarily conserved DNA degradation pathway.  Our study also demonstrates for the first time that mitochondria is important for apoptosis in invertebrates and is a conserved regulator of apoptosis (Abstract and PDF).

 

2) Characterization of the C. elegans mitochondrial cell death pathways

            The findings that apoptotic regulators such as cytochrome c, endoG, and apoptosis-inducing-factor (AIF) are released from mitochondria to mediate different aspects of apoptosis indicate that mitochondria plays an important role in mammalian apoptosis.  A family of proteins containing a unique Bcl-2 homology 3 motif (BH3) are involved in releasing these mitochondrial apoptogenic factors.  The finding that cps-6 encodes a mitochondrial endonuclease prompted us to examine whether there are additional mitochondrial proteins involved in C. elegans apoptosis.  We characterized the C. elegans homologue of AIF, a human mitochondrial oxidoreductase that is released from mitochondria during apoptosis to induce chromosome condensation and fragmentation.  Intriguingly, the oxidoreductase activity of AIF is dispensable for its apoptogenic functions and AIF does not possess a nuclease activity, raising a major question of how AIF induces apoptosis.  We cloned the worm AIF homologue, wah-1, and used RNA interference (RNAi) to study its functions in C. elegans apoptosis.  We found that reduction of the wah-1 activity in C. elegans delays the normal progression of apoptosis, results in accumulation of TUNEL-positive DNA breaks, and enhances the defects of other cell death mutants, indicating that C. elegans AIF does play an important role in regulating C. elegans apoptosis.  Interestingly, wah-1(RNAi) results in cell death phenotypes that are similar to those of the cps-6 mutant and fails to enhance the cps-6 cell death defects, indicating that wah-1 and cps-6 function in the same cell death pathway.  Indeed, we found that WAH-1, which also localizes to C. elegans mitochondria, can associate and cooperate with CPS-6 in vitro to promote DNA degradation.  In vivo, co-expression of wah-1 and cps-6 can synergistically induce cell killing.  These findings indicate that CPS-6/EndoG is likely a target or an effector that WAH-1/AIF interacts with to induce chromosome fragmentation during apoptosis and that CSP-6/EndoG and WAH-1/AIF define a single evolutionarily conserved cell death pathway initiated from mitochondria.  Importantly, WAH-1 can be released from mitochondria by EGL-1, a C. elegans BH-3-domain containing cell death activator, in a manner similar to the release of cytochrome c or EndoG from mitochondria by mammalian BH3-domain containing proteins such as Bid or Bim.  However, this EGL-1-induced release of WAH-1 is dependent on the activity of the CED-3 protease, indicating that worm AIF functions in a caspase-dependent manner.  These observations strongly suggest that the mitochondrial cell death pathway is conserved between nematodes and humans (Abstract and PDF).

 

We are currently focusing on identifying additional mitochondrial factors that are important for the regulation and execution of programmed cell death in C. elegans using both genetic and biochemical approaches.  We are particularly interested in understanding how mitochondrial apoptogenic factors are released from mitochondria to affect various aspects of apoptosis.  We hope to identify most components functioning in C. elegans mitochondrial cell death pathways.

 

 

3) Functional genomic analysis of the apoptotic DNA degradation process in C. elegans

The observations that multiple genes (cps-3, 4, 6, 7, 8, 10, 11, 13, 14 and wah-1) are involved in the apoptotic DNA degradation process in C. elegans indicate that this is likely a rather complicated and tightly regulated process.  To identify all the nucleases involved in C. elegans apoptosis, we conducted a candidate-based, genome-wide RNAi screen to systematically search for genes important for apoptotic DNA degradation in C. elegans.  We used RNAi to individually inhibit the expression of 77 C. elegans genes that encode nucleases or nuclease-related proteins and have identified nine candidate apoptotic nucleases, including two previously known apoptotic nucleases (CPS-6 and NUC-1).  We named these new cell death-related nucleases as CRN nucleases.  Molecular genetic analyses of these crn genes indicate that these nine apoptotic nucleases comprise at least two independent pathways that contribute to cell killing by degrading chromosomal DNA, with cps-6, crn-1, crn-4, crn-5, and crn-7 acting together in one pathway and crn-2/crn-3 in another pathway (Figure 2).  nuc-1 and crn-6 appear to act at later stages of apoptotic DNA degradation.  Interestingly, several crn genes have human homologues that play important roles in RNA processing and splicing, protein folding, DNA replication and damage repair, suggesting that these CRN proteins may play dual roles in both cell survival and cell death.  The identification of seven crn genes will allow systematic deciphering of the mechanisms of apoptotic DNA degradation, which remain a poorly understood biological process (Abstract and PDF).

 

As a first step towards the understanding of how apoptotic nucleases interact to promote apoptotic DNA degradation, we initiated biochemical and mechanistic studies of CRN-1, a homologue of the human Fen-1 endonuclease that plays important roles in DNA replication and repair.  We found that CRN-1 localizes to nuclei and can associate and cooperate with CPS-6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS-6 and both the 5Õ-3Õ exonuclease activity and a novel gap-dependent endonuclease activity of CRN-1.  Our results suggest that CRN-1/FEN-1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis (Abstract and PDF).

 

4) Identification and characterization of genes involved in exposure or recognition of Òeat-meÓ signals during removal of apoptotic cell corpses

            Phagocytosis of apoptotic cells is an integral part of cell death execution and an important event in tissue remodeling, suppression of inflammation, and regulation of immune responses.  During apoptosis, Òeat-meÓ or engulfment signals are exposed or released from the dying cells to trigger the phagocytic events by neighboring cells or macrophages.  Very little is known about what these Òeat-meÓ signals are or how they are exposed or released from the dying cells.  One of the candidate Òeat-meÓ signals is phosphatidylserine (PS), which normally is restricted to the inner leaflet of the plasma membrane but is exposed on the surface of apoptotic cells.  It is unclear what regulates the externalization of PS in apoptotic cells and how phagocytes recognize PS and subsequently initiate the phagocytic events.  From our cps screens, we have identified three genes (cps-12, cps-13 and cps-14) that affect the externalization of PS in apoptotic cells.  Molecular characterization of these three genes will provide important insights on how PS externalization is regulated and executed in apoptotic cells.

 

Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate PS recognition and phagocytosis.  Interestingly, C. elegans contains a gene (F29B9.4) that shares 56% sequence identity with human PSR.  To investigate whether the C. elegans PSR homologue (named psr-1) affects cell corpse engulfment, we isolated a deletion allele (tm469) in the psr-1 locus and analyzed its mutant phenotypes.  We found that the psr-1(tm469) mutation does result in a defect in cell corpse engulfment.  Interestingly, psr-1 appears to act in the same cell corpse engulfment pathway as ced-2, ced-5, ced-10 and ced-12.  Genetic bypass experiments indicate that psr-1 acts upstream of ced-2, ced-5, ced-10 and ced-12 to control cell corpse engulfment.  In vitro C. elegans PSR-1 behaves like human PSR and binds preferentially PS or cells with exposed PS.  Interestingly, the intracellular domain of PSR-1 interacts with CED-5 and CED-12, suggesting that PSR-1 may transduce engulfment signal through CED-5 and CED-12.  Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10 and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis (Abstract and PDF).

 

Related Publications

 

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. (Abstract and PDF).  Nature Reviews Mol. Cell Biol.

 

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.  (Abstract and PDF).  Nature Reviews Molecular Cell Biology (PDF)

 

Parrish, J. and Xue, D. (2003). Functional genomic analysis of apoptotic DNA degradation in C. elegans.  Mol. Cell 11, 987-996.  (Abstract and PDF)

 

Parrish, J., Yang, C.L., Shen, B.H., and Xue, D. (2003). CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptoticDNA degradation.  EMBO. J. 22, 3451-3460.  (Abstract and PDF)

 

Wang, X.C., Wu, Y.C., Fadok, V., Lee, M.C., Gengyo-Ando, K., Cheng, L.C., Ledwich, D., Hsu, P.K., Chen, J.Y., Chou, B.K., Henson, P., Mitani, S., and Xue, D. (2003).  Cell Corpse Engulfment Mediated by C. elegans Phosphatidylserine Receptor Through CED-5 and CED-12. Science 302, 1563-1566. (Abstract and PDF).  Science Perspectives (PDF)

 

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. (Abstract and PDF). Nature Reviews Molecular Cell Biology (PDF)

 

Darland-Ransom, M., Wang, X.C., Sun, C.L., Mapes, J., Gengyo-Ando, K., Mitani, S. and Xue, D. (2008). Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry. Science 320, 528-531. (Abstract and PDF).  Science Perspectives (PDF)

 

 

 

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Abstracts on the studies of targets of cell death proteases

The abstract of a talk presented at the 12th International C. elegans meeting (1999)

Identification and Characterization of Downstream Targets of the Cell-Death Protease CED-3

DA Ledwich, CE Duffy, CK Lau, HE Metters, PT Huynh, D Xue, CB 347, MCDB, Univ. of Colo., Boulder, CO 80309

Analysis of downstream pathways of important enzymatic biomolecules (e.g., kinases and proteases) which have multiple substrates has always been a difficult challenge. Targets of these enzymes could be difficult to identify through conventional genetic screens since elimination of one of the multiple targets of an enzyme may fail to cause any visible phenotype that can be scored in genetic screens. Here we report a novel genetic screen that will allow us to identify most of the downstream targets of the CED-3 death protease.

The cell-death gene ced-3 encodes a cysteine protease that executes programmed cell death by cleaving critical protease targets. To identify the downstream targets and pathways that mediate CED-3 cell-killing activity, we carried out a novel and sensitive genetic screen to isolate mutations that partially suppress or delay cell death caused by constitutively activated CED-3 death protease. In this screen, we co-expressed CED-3 with GFP in six touch receptor neurons and used GFP fluorescence as a sensitive cell-existence marker to detect the presence of touch receptor neurons whose death is either partially suppressed or delayed by mutations. So far, we have isolated more than sixty such mutations. Five of them are alleles of the ced-1 gene, suggesting that it is a plausible strategy to identify genes that act downstream of ced-3 (ced-1 acts downstream of ced-3 to mediate cell corpse engulfment). The other mutations define at least six new genes, which we named cps genes (CED-3 protease suppressors). Importantly, mutations in these cps genes not only affect CED-3-mediated ectopic cell-death but also affect normal programmed cell death in nematodes. Specifically, mutations in cps-1 and cps-2 weakly suppress general cell death, while mutations in cps-3, cps-4, cps-5and cps-6 act like ced-8 mutations by causing the delay of cell-killing (1). Epistasis analysis suggests that six cps genes act downstream of ced-3 but upstream of the engulfment ced genes. This novel genetic screen should help us identify most of the components and pathways that act downstream of the CED-3 death protease to execute cell death, which currently is the missing link of the cell death pathway.

 

The abstract of a talk presented at the ÒProgrammed cell deathÓ symposium held at CSHL (2001)

A Genetic Pathway Involved In Apoptotic DNA Degradation In Nematodes

Jay Parrish1, Kristina Klotz1, Duncan Ledwich1, Hye-Seong Park1, Lily Li2, Xiadong Wang2 and Ding Xue1

 

1Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO  80309, USA

2HHMI and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX  75390, USA

 

Programmed cell death (apoptosis) is a tightly regulated cell disassembly process which is characterized by shrinkage and fragmentation of both dying cells and their nuclei as well as fragmentation of chromosomal DNA into internucleosomal repeats.  The execution of these systematic and orderly cell disassembly processes is regulated by apoptotic caspases, which trigger these events by cleaving critical protease targets, most of which remain to be identified.  To identify genetic components that act downstream of, or in parallel to, the C. elegans caspase CED-3, we have carried out a sensitized GFP-based genetic screen to isolate suppressors of a constitutively activated CED-3 mutant.  Using this screen we have isolated 64 suppressor mutations that define at least 8 new cell death genes, which we named cps genes (CED-3 protease suppressors).  We found that loss-of-function mutations in cps genes result in either a partial suppression of programmed cell death (cps-1, 2) or a delay in the timing of cell death (cps-3 to cps-8).

            We have focused on studying the possible role of the cps genes in the apoptotic DNA degradation pathway in C. elegans.  Using the TUNEL assay which labels 3Õ-OH DNA breaks generated during apoptosis (Wu et al., 2000), we found that mutations in at least five cps genes result in accumulation of TUNEL-positive staining in mutant embryos, indicating that these mutants are defective in resolving TUNEL-positive DNA ends.  Further genetic analysis indicates that these five genes are likely to function in the same apoptotic DNA degradation pathway.  We have characterized one of these genes, cps-6, in detail and have found that it encodes a homologue of mammalian mitochondrial endonuclease G, which is implicated in mediating apoptotic DNA degradation in mammals (Li et al., 2001).  Furthermore, we showed that CPS-6, like its mammalian counterpart, also localizes to mitochondria in C. elegans.  CPS-6 is thus 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.  Our studies also provide the first genetic evidence that the apoptotic DNA fragmentation process is important for proper progression of apoptosis.

We are now investigating how CPS-6 is released from mitochondria and activated during apoptosis to mediate the nuclear DNA fragmentation process.  We are examining how the activity of cps-6 is regulated by other cps genes or by previously identified cell death genes.  Additionally, we are in the process of cloning the other cps genes to further understand their roles and functional relationships with cps-6 in mediating apoptotic DNA degradation.  Finally, we are using a variety of genetic and cell biological techniques to determine when and where cps-6 acts during the apoptotic DNA degradation process.  These studies should reveal the molecular and biochemical mechanisms that regulate apoptotic DNA fragmentation.

 

The abstract of a talk presented at the 13th International C. elegans meeting (2001)

A Genetic Pathway Involved In Apoptotic DNA Degradation In Nematodes

Jay Z Parrish, Hye-Seong Park, Duncan A. Ledwich, Kristina Klotz, Helen E. Metters, and Ding Xue, CB 347, MCDB, Univ. of Colo., Boulder, CO 80309

Programmed cell death (apoptosis) is a tightly regulated cell disassembly process, which includes shrinkage and fragmentation of both dying cells and their nuclei as well as fragmentation of chromosomal DNA into internucleosomal repeats, a biochemical hallmark of apoptosis.  The execution of these systematic and orderly cell disassembly processes is regulated by apoptotic caspases, which trigger these events by cleaving critical protease targets, most of which remain to be identified.  To identify genetic components that act downstream of, or in parallel to, the C. elegans caspase CED-3, we have designed and carried out a sensitized GFP-based genetic screen to isolate suppressors of a constitutively activated CED-3 mutant (Ledwich et al., 1999).  Using this screen we have isolated 64 suppressor mutations which define at least 8 new cell death genes, which we named cps genes (CED-3 protease suppressors).  Further genetic and phenotypic analyses of cps mutants indicate that loss-of-function mutations in cps genes result in either a partial suppression of programmed cell death (cps-1, 2) or a delay in the timing of programmed cell death (cps-3 to cps-8).

We have focused on studying the possible role of the cps genes in the apoptotic DNA degradation pathway in C. elegans.  Using the TUNEL assay which labels 3Õ-OH DNA breaks generated during apoptosis (Wu et al., 2000), we found that mutations in four cps genes result in accumulation of TUNEL-positive staining in mutant embryos, indicating that these mutants are defective in resolving TUNEL-positive DNA ends.  Further genetic analysis indicates that these four genes are likely to function in the same apoptotic DNA degradation pathway.  We have characterized one of these genes, cps-6, in detail and have found that it encodes a specific C. elegans mitochondrial endonuclease, whose role in apoptosis appears to be evolutionarily conserved. CPS-6 is thus the first mitochondrial protein identified to be directly involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.  Our studies also provide the first genetic evidence that the apoptotic DNA fragmentation process is important for proper progression of apoptosis.  We are now investigating how CPS-6 is released from mitochondria and activated during apoptosis to mediate the nuclear DNA fragmentation process.  We are also examining how the activity of cps-6 is regulated by other cps genes or by previously identified cell death genes.  Finally, we are in the process of cloning three other cps genes in the same pathway to further understand their roles and functions in mediating apoptotic DNA degradation.  These studies should reveal the molecular and biochemical mechanisms that regulate the apoptotic DNA fragmentation process in C. elegans and in general.

 

The abstract of a poster presented at the 13th International C. elegans meeting (2001)

Identification and characterization of cps-4 and cps-5

Hye-Seong Park, David Kokel, Duncan Ledwich, Tim Greiger, and Ding Xue, Department of MCD Biology, University of Colorado, Boulder, CO 80309 USA

Programmed cell death (apoptosis) is a complex and tightly controlled process that is vital for the proper development of an organism as well as for maintaining its homeostasis. ced-3, a key player in the execution of programmed cell death in C. elegans, encodes a member of the caspase family of cysteine proteases. One particularly important area that has not been studied is the in vivo targets of the death caspases. In order to reveal downstream targets of CED-3, our lab developed a novel and sensitized genetic screen to isolate mutations that partially suppress or delay cell death caused by constitutively activated CED-3 death protease. In this screen, at least eight new genes (cps-1,-2,-3,É,-8; CED-3 protease suppressors) have been identified. Here we report the characterization of the cps-4 and cps-5 genes which appear to function in interesting yet different ways to affect apoptosis.

To study how cps-4 and cps-5 affect programmed cell death in nematodes, we performed time-course analyses of the appearance of embryonic cell corpses in cps-4 and cps-5 mutants. We found that both cps-4 and cps-5 mutants display a delay of cell death phenotype: the peak of cell corpses is shifted from the bean/comma embryonic stage (seen in wild type animals) to the 2-fold embryonic stage in cps-4 and cps-5 mutants. These phenotypes are similar but weaker than the delay-of-cell-death phenotype displayed by another cell death mutant, ced-8, in which the peak of cell corpses is found in late embryonic stage (late three-fold embryonic stage). We constructed double and triple mutants among cps-4, cps-5, ced-8 and other cps genes and found that cps-4, cps-5, and ced-8 can significantly enhance one another's phenotype in delaying cell death. In addition, cps-5 but not cps-4 can also enhance the delay-of-cell-death phenotype of cps-6, which is involved in apoptotic DNA degradation and encodes an endonuclease (please see the abstract by Parrish et. al.). These results suggest that cps-4 and cps-6 may function in the same pathway while cps-5 and ced-8 may function in different pathways. Consistent with this hypothesis, we found that cps-4 mutants contained significantly higher number of TUNEL-positive cells than that of wild-type animals, while the cps-5 mutant has a similar number of TUNEL-positive cells as the wild-type animals.

We mapped cps-4 to linkage group I and cps-5 to linkage group III and are in the process of fine mapping and cloning these two genes.

 

The abstract of a poster presented at the 13th International C. elegans meeting (2001)

Study of the function of phosphatidylserine receptor in C.elegans

Xiaochen Wang1, Duncan Ledwich1, Valerie A. Fadok2, Ding Xue1

1Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO80309

2Dept. Pediatrics, National Jewish Medical and Research Center, Denver CO 80206

The recognition and clearance of apoptotic cells is an important step during apoptosis. In this process, it has been observed that apoptotic cells lose phospholipid asymmetry and expose phosphatidyserine (PS), an anionic phospholipid, on the outer surface of the plasma membrane. The exposed PS has been proposed to serve as a recognition marker for engulfing cells. How the exposed phosphatidylserine is recognized and mediates the corpse engulfment process is still largely unknown. Recently, a phosphatidylserine receptor (PSR) that appears to mediate specific recognition of PS on apoptotic cells by phagocytes has been identified in humans1. It is expressed on the surface of macrophages, fibroblasts and epithelial cells. When transfected into cultured cells, the PSR allows cultured B and T lymphocytes to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. This phosphatidylserine receptor is highly conserved throughout evolution. We have noted the presence of a homologous gene in C.elegans that shares high sequence homology with human PSR (46% identity).

In order to investigate the possible role of the putative worm PSR homolog in the engulfment of dying cells during C. elegans programmed cell death, we are currently performing RNAi experiments and looking for deletions in the PSR homolog locus to examine the loss-of-function phenotypes of worm PSR. We have made several PSR::GFP fusions and are in the process of determining its expression pattern and localization in nematodes. These experiments will help us better understand the function of PSR in nematodes and also will likely provide additional important information about the mechanisms of cell corpse recognition and clearance in C. elegans.

1.Fadok, V.A. etal. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405,85-90(2000).

 

The abstract of a poster presented at the 14th International C. elegans meeting (2003)

Molecular genetic characterization of cps-1, a CED-3 suppressor

 

Dave Kokel, Marcela Valencia, Corine Lau, Ding Xue. MCD Biology, University of Colorado, Boulder, CO.

  

Programmed cell death (PCD) is a tightly regulated cell disassembly process in which cells undergo stereotypical morphological changes including cytoplasmic shrinkage, chromatin condensation and fragmentation, and cell corpse engulfment. The CED-3 caspase likely causes PCD by activating multiple cell disassembly processes. However, the identities of the molecules that act in these cell disassembly pathways are mostly unknown. In order to identify genes that act downstream of ced-3 to promote PCD, we have designed and carried out a genetic screen to isolate suppressors of an activated CED-3 protease. From this screen we have identified more than ten ced-3 protease suppressor (cps) genes. Three partial loss-of-function mutations in one of these genes, cps-1, appear to reduce the number of programmed cell deaths in C. elegans.

 

Results from three different cell death assays suggest that PCD is partially suppressed in cps-1 mutants. First, neurons that are induced to undergo cell death by ectopic expression of activated CED-3 survive more frequently in cps-1 mutants than in wild type animals. Secondly, fewer cell corpses are seen in cps-1 mutant embryos than in wild type embryos. Lastly, cps-1 mutations can enhance the cell killing defects of other weak cell death mutants. For example, a cps-1 mutation (sm24) significantly increases the number of undead cells in the anterior pharynx of a weak ced-3 mutant (n2438). We have mapped cps-1 to a small region of chromosome I and are in the process of cloning this gene.

 

 

The abstract of a poster presented at the 14th International C. elegans meeting (2003)

 

Functional genomic analysis of apoptotic DNA degradation in C. elegans.

 

Jay Z. Parrish, Nathan D. Camp, Ding Xue. MCDB, University of Colorado, Boulder, CO.

 

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Classical genetic screens have been ineffective at identifying the components of the DNA degradation machinery and mutant alleles in known apoptotic nucleases do not display easily detectable phenotypes. In order to overcome this problem, we have used the TUNEL assay to conduct an RNAi-based functional genomic screen to identify additional nucleases involved in apoptotic DNA degradation. From an initial screen of 77 open reading frames (ORFs) encoding C. elegans nucleases, cyclophilins and topoisomerases, we have isolated nine cell death-related nucleases (crn genes), two of which (cps-6 and nuc-1) were previously known to function in apoptotic DNA degradation. Two genes, crn-6 and cyp-13, encode homologues of mammalian genes previously implicated in apoptotic DNA degradation (DNase II and cyclophilin family proteins, respectively), demonstrating that these genes likely play a conserved role in DNA degradation. Genetic and phenotypic analyses suggest that these crn genes likely function in two distinct, partially independent pathways to mediate apoptotic DNA degradation, with cps-6, crn-1, crn-4, crn-5, and cyp-13 functioning in one pathway and crn-2 and crn-3 acting in the other pathway. Interestingly, five of the nucleases (including CPS-6) that act in the same pathway and at least one non-nuclease component, WAH-1 (worm apoptosis-inducing factor homologue), appear to interact with one another in vitro and may assemble into a protein complex to mediate apoptotic DNA degradation in vivo. We have named this complex the degradeosome.

   

Currently, we are expanding the screen to include recently annotated nucleases and nuclease related genes to identify the nuclease(s) functioning upstream of cps-6 to initiate apoptotic DNA degradation and to identify additional components of the degradeosome. Furthermore, we are conducting a genetic suppressor screen to isolate mutations that suppress crn-2/3 or cps-6-induced ectopic cell killing to identify additional nuclease and non-nuclease components that participate in two different DNA degradation pathways. Finally, we are characterizing several cps (ced-3 protease suppressor) mutants that are also defective in apoptotic DNA degradation. Taken together, these studies should advance our understanding of the