Melendez A et al. (2009) WormBook "Autophagy in C. elegans."

Levine B, Melendez A

[WormBook, 2009]

Autophagy is a ubiquitous cellular process responsible for the bulk degradation of cytoplasmic components through an autophagosomal-lysosomal pathway. Genetic screens, primarily in S. cerevisiae, have identified numerous genes that are essential for autophagy. Many of these genes have orthologs in higher eukaryotes, including C. elegans, Drosophila, and mammals. Gene knockdown/knockout studies in C. elegans have been useful to probe the functions of autophagy in an intact multicellular organism that undergoes development to produce different cell types. This review summarizes important themes that have emerged regarding the roles of autophagy in C. elegans in adaptation to stress, aging, normal reproductive growth, cell death, cell growth control, neural synaptic clustering, and the degradation of aggregate-prone proteins.

Melendez A et al. (1996) East Coast Worm Meeting "lin-13 encodes a zinc finger protein"

Melendez A, Greenwald IS

[East Coast Worm Meeting, 1996]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are Multivulva (Muv) and sterile. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. lin-13 maps very close and to the left of mab-5 (M. Costa and C. Kenyon, personal communication). Pools of cosmids for the implicated genomic region were tested in germline transformation experiments, and cosmids C03B8 and K04F4 can rescue both the sterility and Muv phenotype of lin-13 (G. Kao, A. Mele'ndez and I. Greenwald, unpublished observations). A 16-kb genomic (Mlu I) fragment rescued the Muv and sterility phenotypes of lin-13 mutants. The genomic DNA corresponding to the lin-13 rescuing Mlu I fragment contains at least two predicted open reading frames: a protein containing many zinc finger motifs and a protein with ankyrin repeats (A. Coulson, J. E. Sulston and R. H. Waterson, personal communication). We found that a 12-kb (BstE II-Mlu I) fragment, excluding the 5' end of the predicted zinc finger containing protein, no longer rescued. By contrast a 12-kb (Mlu I-Sal I) fragment that included the predicted zinc finger protein and eliminated most of the genomic region predicted to encode the ankyrin repeat protein still rescued. We have also introduced a frameshift mutation into the predicted zinc finger protein coding region of the 16-kb Mlu I genomic fragment and it no longer rescued. All of these results suggest that lin-13 encodes a zinc finger containing protein. lin-13 has been grouped with the SynMuv genes (Ferguson et al. 1987). Studies of SynMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We therefore plan to determine the cellular focus of lin-13 activity by using reporter genes and genetic mosaic analysis.

Melendez A et al. (2008) Methods Enzymol "Monitoring the role of autophagy in C. elegans aging."

Hall DH, Hansen M, Melendez A

[Methods Enzymol, 2008]

Autophagy plays crucial roles in many biological processes, and recent research points to a possibly conserved role for autophagy in the process of organismal aging. Experiments in the nematode C. elegans suggest that autophagy may be required specifically for longevity pathways that are regulated by environmental signals. Known longevity genes can be assigned to four major longevity pathways/processes: insulin/IGF-1 signaling, dietary restriction, protein translation, and mitochondrial respiration. Of these, reduced insulin/IGF-1 signaling and dietary restriction, but not protein translation inhibition, appear to rely on autophagy to increase life span. Multiple experimental approaches have been used to study autophagy in the context of aging in C. elegans. This chapter describes techniques used to address the link between aging and autophagy in C. elegans. Specifically, we summarize how to examine organismal life span in various longevity mutants and how to visually detect autophagy and autolysosomal formation in C. elegans.

Melendez A et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON LIN-13. A ZINC-FINGER PROTEIN THAT ACTS IN VULVAL DEVELOPMENT"

Greenwald IS, Melendez A

[International C. elegans Meeting, 1997]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are sterile and display a Multivulva phenotype. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. Along with Gautam Kao, we rescued the Muv and sterility phenotypes of lin-13 with cosmids CO3B8 and KO4F4. We have narrowed the rescuing activity to a 12-kb (MluI-SalI) genomic fragment that encodes a predicted zinc finger protein, and a frameshift mutation introduced into this coding region abolishes rescuing activity. We have isolated cDNAs corresponding to this genomic region and have started characterizing them. The lin-13 transcript appears to be SL1-spliced and of approximately 7-kb in length, encoding a large protein of about 2,100 amino acids with 14 zinc-fingers of the C-X(2-4)-C-X(3)-F-X(5)-L-X(2)-H-X(3)-H class. lin-13 has been grouped with the synMuv genes, which define two functionally redundant pathways that negatively regulate vulval development (Ferguson et al. 1987; Ferguson and Horvitz, 1989). Studies of synMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We have begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development (lin-13 reporter constructs suggest that it is expressed in both hyp7 and the VPCs). We are also screening for lin-13 null alleles to understand further the role of lin-13 in development.

Alicia Melendez et al. (2001) International C. elegans Meeting "What is the cellular focus of SynMuv genes?"

Iva Greenwald, Alicia Melendez

[International C. elegans Meeting, 2001]

The synthetic Multivulva (synMuv) genes comprise two classes of genes, Class A and Class B, that act in the determination of vulval precursor cell (VPC) fates (Ferguson, E.L. and Horvitz, H.R.,1985,1989). A synthetic Multivulva phenotype is produced by two mutations, one in each of the two classes of genes. Mutations in one or more class A genes or one or more class B genes do not produce a Multivulva phenotype. It has been proposed that the class A and class B genes define two functionally redundant pathways that act to specify the non-vulval fate of the vulval precursor cells. We have previously shown that lin-13 behaves as a Class B SynMuv gene, a class that also includes genes encoding the tumor suppressor Rb and RbAp48, a protein that binds Rb. We have also shown that LIN-13 is a nuclear protein that contains multiple zinc fingers and a motif, LXCXE, that has been implicated in Rb binding. These results together suggested a role for LIN-13 in Rb-mediated repression of vulval fates. A Multivulva phenotype can in principle result from activation of a pathway or process that promotes vulval fates, or the inhibition of a pathway or process that represses vulval fates. Genes that influence this process can in principle act in an external signalling cell or in the VPCs. There have been two views to date. One view is that SynMuv genes act in an external signalling cell, hyp7, to control expression of a factor that antagonizes the effects of the inductive signal or otherwise to promote nonvulval fates (Herman and Hedgecock 1990). The other view is that SynMuv genes act in the VPCs to repress transcription of genes that are involved in vulval differentation (Lu and Horvitz 1998). The proposal that SynMuv genes act in hyp7 was based on genetic mosaic studies of a lin-15 mutation that concomitantly removes both Class A and Class B activities (Herman and Hedgecock 1990), and was supported by mosaic analysis of the Class B SynMuv gene lin-37 (Hedgecock and Herman 1995). Our findings that lin-13::lacZ and LIN-13::GFP are consistently expressed in hyp7 and are generally undetectable in VPCs at the time of VPC specification has been consistent with this view. If SynMuv genes act in hyp7 and not in the VPCs, they may promote the expression in hyp7 of an inhibitory factor that represses vulval fates. As it remains possible that some SynMuv genes act in hyp7 while others act in the VPCs, and as expression data can be misleading as to cellular focus, we have undertaken genetic mosaic analyses of various SynMuv genes. We will report on our progress and plans concerning mosaic analysis of lin-13 and lin-35 and expression of SynMuv genes in specific cells.

Melendez A et al. (2003) Science "Autophagy genes are essential for dauer development and life-span extension in C. elegans."

Levine B, Seaman M, Melendez A, Hall DH, Talloczy Z, Eskelinen EL

[Science, 2003]

Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1 the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1 is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.

A Melendez et al. (1999) International C. elegans Meeting "LIN-13, a zinc-finger protein that acts in vulval development"

A Melendez, IS Greenwald

[International C. elegans Meeting, 1999]

The synthetic Multivulva (synMuv) genes comprise two classes of genes that act in the determination of vulval precursor cell (VPC) fates (1,2). Under certain conditions, alleles of lin-13 display some of the genetic properties of a Class B SynMuv gene (2; our observations, which we will present). We have found that lin-13 encodes a large nuclear protein of 2,249 amino acids with 14 zinc-fingers of the C2-H2 class. Both of the existing lin-13 alleles are nonsense mutations. lin-13(n387) is a TCA to TGA (S525stop) change that disturbs the first zinc-finger and would encode a predicted protein of 524 amino acids. lin-13(n388) is a CGA to TGA (R857stop) change right after the second zinc finger and would encode a predicted protein of 856 amino acids. Thus, both nonsense mutations would result in greatly truncated LIN-13 protein products. The molecular nature of the lin-13 alleles was surprising, since Ferguson and Horvitz (1) showed that lin-13 (n387)/nDf16 is lethal which suggested that lin-13(n387) was hypomorphic, and the lin-13 null phenotype is zygotic lethality. To explore the issue of the lin-13 null phenotype, we used RNAi (3). We assayed the phenotype of the progeny of N2 hermaphrodites injected with dsRNA derived from lin-13 cDNA genomic clones. Injected mothers were incubated at 15degC or at 25degC. Progeny raised at 25degC were Muv and sterile, whereas progeny raised at 15degC were sterile but not Muv. Thus, the phenotypes caused by RNAi resemble those caused by the existing lin-13 alleles, and raise the distinct possibility that the null phenotype of lin-13 is the heat sensitive phenotype displayed by the two existing alleles. We have also begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development ( lin-13 reporter constructs suggest that it is expressed mainly in hyp7 and possibly in the VPCs). We will report on our progress and we will also speculate about the connection between lin-13 and the class B synMuv gene lin-35Rb . 1. Ferguson, E.L. and Horvitz, H.R. (1985) Genetics 110, 17-72. 2. Ferguson, E.L. and Horvitz, H.R. (1989) Genetics 123, 109-121. 3. Fire, A., et al. (1998) Nature 391, 806-811.

Alicia Melendez et al. (2005) International Worm Meeting "An essential role for the autophagy gene bec-1 in C. elegans development"

Beth Levine, Alicia Melendez

[International Worm Meeting, 2005]

Autophagy is the major cellular pathway for degrading long-lived proteins and cytoplasmic organelles. Although it contributes to routine turnover of cytoplasmic components, it can be induced by a number of stimuli, including nutrient starvation, hypoxia, intracellular stress, hormones, and various developmental signals. We are analyzing the role of autophagy genes in C. elegans development, by studying the function of the bec-1 gene. The human ortholog of bec-1, beclin 1 is the first identified mammalian gene to mediate autophagy and also has tumor suppressor and antiviral function (Liang et al., 1999). In yeast, the bec-1 gene was isolated as an autophagy defective mutation, atg6, or as a vacuolar protein sorting mutation, vps30. We have previously shown that C. elegans bec-1, like human Beclin 1 can rescue autophagy, but not vacuolar protein sorting in an autophagy-defective yeast atg6/vps30 mutant. Furthermore, we have shown that bec-1 and other autophagy genes are required for dauer development and longevity (Melendez et al., 2003). Analysis of bec-1(ok691), a molecular null allele (Gene Knockout consortium), suggests an essential role for bec-1 in early development. Removing bec-1 activity zygotically leads to arrest at various developmental stages with increasing vacuolization and uncoordinated phenotypes. Occasionally, mutant animals reach adulthood, yet they are invariably sterile. Removing both maternal and zygotic bec-1 activity results in early embryonic lethality. We are currently defining the cellular focus of the bec-1 lethal phenotype by mosaic analysis and cell specific rescue. To identify genes that genetically interact with bec-1, we are conducting a screen to isolate suppressors of the bec-1 lethal and/or sterility phenotype. bec-1(0) hermaphrodites transgenic for a rescuing bec-1(+) array and a GFP transformation marker (marking the rescuing array) were mutagenized with EMS and the progeny screened for the presence of non-GFP expressing fertile adults. These adults are still bec-1(0) homozygous and should carry an extragenic suppressor of the bec-1 lethality. In a small pilot screen, we have isolated two suppressors at a frequency of approximately 1/1000 haploid genomes. In a complementary approach, we are also performing a screen for bec-1 suppressors by using transposon mediated mutagenesis (Mos1)(Bessereau et al., 2001). Genetic characterization of the two new suppressors is in progress. Since autophagy has been linked to tumor suppression, the identification of bec-1 interacting genes may identify new candidate genes involved in cancer biology. Molecular characterization of these genes will lead to a greater understanding of bec-1 function in development and disease.

Melendez A et al. (2000) Genetics "Caenorhabditis elegans lin-13, a member of the LIN-35 Rb class of genes involved in vulval development, encodes a protein with zinc fingers and an LXCXE motif."

Greenwald IS, Melendez A

[Genetics, 2000]

The SynMuv genes appear to be involved in providing a signal that inhibits vulval precursor cells from adopting vulval fates in Caenorhabditis elegans. One group of SynMuv genes, termed class B, includes genes encoding proteins related to the tumor suppressor Rb and RbAp48, a protein that binds Rb. Here, we provide genetic evidence that lin-13 behaves as a class B SynMuv gene. We show that null alleles of lin-13 are temperature sensitive and maternally rescued, resulting in phenotypes ranging in severity from L2 arrest (when both maternal and zygotic activities are removed at 25 degrees ), to sterile Multivulva (when only zygotic activity is removed at 25 degrees ), to sterile non-Multivulva (when both maternal and zygotic activities are removed at 15 degrees ), to wild-type/class B SynMuv (when only zygotic activity is removed at 15 degrees ). We also show that LIN-13 is a nuclear protein that contains multiple zinc fingers and a motif, LXCXE, that has been implicated in Rb binding. These results together suggest a role for LIN-13 in Rb-mediated repression of vulval fates.

Alicia Melendez et al. (2001) International C. elegans Meeting "Analysis of the C. elegans homolog of the mammalian autophagy gene beclin 1"

Alicia Melendez, David H Hall, Beth Levine

[International C. elegans Meeting, 2001]

Autophagy is the bulk degradation of cytoplasmic components through an autophagosomic-lysosomal pathway. Under stress or nutrient starvation, mammalian cells as well as yeast cells undergo major degradation of proteins and remodelling of cellular compartments. Autophagy is essential for differentiation, survival during nutrient deprivation, and normal growth control; however, nothing is known about autophagy in the development of multicellular organisms. We are analyzing the role of evolutionarily conserved autophagy genes in C. elegans development, by studying the function of the worm beclin1 homolog. The human beclin 1 gene is the first identified mammalian gene to mediate autophagy and also has tumour suppresor and antiviral function (Liang et al., 1999). In yeast, the beclin1 gene was isolated as an autophagy defective mutation, Apg6 , or as a vacuolar protein sorting mutation, Vps11 . Mammalian Beclin 1 promotes autophagy, but not vacuolar protein sorting in autophagy-defective yeast with a targeted disruption of the yeast ortholog, APG6/VPS30 (Liang et al., 1999). Human Beclin1 shares 28% amino acid with the C. elegans Beclin1 (T19E7.3; C. elegans genome sequencing project). We have inactivated the C. elegans beclin1 homologue by injecting T19E7.3 dsRNA or by soaking N2 worms with T19E7.3 dsRNA. beclin1 RNAi-soaked N2 worms arrested at the L1 stage, whereas, the progeny of the injected beclin1 RNAi N2worms reached adulthood with varying defects in the intestine, including the accumulation of large vacuolar structures in the intestine. Ce beclin1 RNAi also decreases the endocytosis of a YP170(yolk protein) ::GFP fusion by oocytes, an assay that has been used to isolate mutants in receptor mediated endocytosis (Grant and Hirsh, 1999). As autophagy is required for survival under stress or nutrient starvation conditions, we hypothesized that autophagy might be required for dauer formation or survival of the dauer larva. To investigate this hypothesis, we injected daf-2 mutants with Ce beclin1 dsRNA. daf-2 mutants are dauer constitutive when raised at the restricted temperature and have an increased lifespan at the permissive temperature (Riddle, 1997). Ce beclin 1 (RNAi); daf-2 double mutants arrested at different stages of dauer formation when grown at the restrictive temperature. Survival of the daf-2 dauers was also affected after beclin1 RNAi. We are now using electron microscopy to determine if autophagy is increased upon dauer formation, and if autophagy is affected in the Ce beclin 1 RNAi mutants. In summary, Ce beclin 1 RNAi-soaked N2 worms arrest at the L1 stage and display intestinal defects, and the Ce beclin 1 RNAi-injected daf-2 worms arrest at different stages of dauer formation. While the molecular basis of these phenotypes is not yet known, these observations suggest a possible role for beclin 1 -mediated autophagy in C. elegans development and in the survival or formation of the C. elegans dauer stage larvae. B. Grant and D. Hirsh, 1999. Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 1999 Dec;10(12):4311-26. X. H. Liang et al., 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999 Dec 9;402(6762):672-6. D. L. Riddle, in Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), pp. 739-768.