Hinrich Schulenburg et al. (2005) International Worm Meeting "Insulin-like signalling links immunity and behavioural defence against pathogens"

Martin Hasshoff, Barbara Hasert, Daniela Tonn, Hinrich Schulenburg

[International Worm Meeting, 2005]

The behavioural avoidance of pathogens minimizes both the inflicted damage and the costs imposed by mounting an immune response required for fighting an infection. Such a strategy should be highly economic and widespread among animals, yet only little is currently known about its occurrence and the underlying mechanisms. In this study, we explore the genetics of pathogen avoidance behaviour in C. elegans. We show that two distinct behavioural defences are mediated through the insulin-like receptor pathway. Since this pathway also contributes to immunity, our study provides the first evidence for a genetic link between physiological and behavioural defences. Our results highlight the importance of insulin signalling as a major life history switch, which determines the optimal life history strategy in response to environmental cues.

Rebecca Schulte et al. (2008) Development & Evolution Meeting "48 Generations of Real-Time Coevolution between C. elegans and its Microparasite Bacillus thuringiensis"

Rebecca Schulte, Carsten Makus, Nico Michiels, Barbara Hasert, Schulenburg Hinrich

[Development & Evolution Meeting, 2008]

Parasites represent a major threat to all organisms resulting in selection for the evolution of host counter-mechanisms. In turn, increased host defenses select for altered parasite pathogenicity, potentially leading to a coevolutionary arms race. Thus, parasite-host coevolution should be characterized by highly dynamic changes over time at both the phenotypic as well as the genetic level. Although the importance of host-parasite coevolution has long been recognized theoretically, conclusive empirical evidence is still extremely rare. We performed a laboratory-based selection experiment in which C. elegans coevolved with its microparasite Bacillus thuringiensis for 48 host generations. Coevolution was contrasted with control conditions where either the host or the parasite was allowed to adapt to the environmental conditions of the experiment. Our results highlight the multifaceted consequences of host-parasite coevolution, including reciprocal changes in resistance and pathogenicity, associated fitness costs, and increased rates of genetic change and recombination (Red Queen dynamics). Taken together, the results emphasize the importance of these coevolutionary interactions as an extremely potent evolutionary force.

Rebecca Schulte et al. (2008) Development & Evolution Meeting "Host-parasite coevolution: Is local adaptation an inevitable outcome?"

Rebecca Schulte, Barbara Hasert, Carsten Makus, Hinrich Schulenburg, Nico Michiels

[Development & Evolution Meeting, 2008]

A host is locally adapted to its parasite if it shows higher resistance to antagonists from the same location than to those from different locations. Similarly, parasites are locally adapted if they are better able to infect co-existing hosts than those from somewhere else. Such host-parasite local adaptation is believed to result from coevolutionary interactions between the counterparts, depending on the exact nature of the interaction as well as various population genetic parameters (e.g. differential migration rates of host and parasite). Its observation in natural populations is widely used as indirect evidence of a past history of coevolution. In the current study, we evaluated the appropriateness of this assumption. For this purpose, we examined C. elegans hosts and their Bacillus thuringiensis microparasites from a laboratory-based evolution experiment. Using this material, we compared the extent of local adaptation between (i) hosts and parasites, which evolved in the presence of the counterpart, and (ii) hosts and parasites, which never encountered the antagonist during experimental evolution. Our results highlight that local adaptation is a common outcome of coevolution. However, at the same time, it was also observed for host-parasite combinations without a shared history, suggesting that it can also be produced by chance. Consequently, the pattern of local adaptation may only be indicative but not sufficient as proof of previous coevolutionary interactions.

Koehler, Fabian et al. (2015) International Worm Meeting "The loss of mma-1 LRPPRC function induces the mitochondrial unfolded protein response."

Koehler, Fabian, Mueller-Rischart, Kathrin, Rolland, Stephane, Conradt, Barbara

[International Worm Meeting, 2015]

In a genetic screen for Caenorhabditis elegans mutants with abnormal mitochondrial morphology, we identified mma-1 (mitochondrial morphology abnormal-1), a C. elegans homolog of the French Canadian Leigh Syndrome gene LRPPRC (leucine-rich pentatricopeptide repeat containing). Inactivation of mma-1 in C. elegans or LRPPRC in mammalian cells results in a decrease in the production of mitochondria-encoded subunits of cytochrome c oxidase (COX) and consequently a reduction in COX activity. We have previously shown that in this context, mitochondria form a hyperfused network that transiently maintains cellular ATP levels (Rolland et al, 2013). We now demonstrate that the inactivation of mma-1 LRPPRC also leads to an imbalance between mitochondria- and nuclear-encoded COX subunits, which triggers a conserved mitochondrial unfolded protein response (UPRmt). We also show that the activation of UPRmt leads to the restoration of mitochondrial proteostasis. Finally, we present evidence that UPRmt and the mitochondrial hyperfusion response are coordinated but mediated by genetically distinct pathways. We propose that in the context of mma-1 LRPPRC knock-down, mitochondrial hyperfusion helps to transiently maintain cellular ATP levels enabling UPRmt to restore mitochondrial proteostasis.Rolland, S.G., Motori, E., Memar, N., Hench, J., Frank, S., Winklhofer, K.F. , and Conradt, B. (2013). Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion. PNAS 110, E2967-2976.Barbara Conradt and Stephane Rolland are co-corresponding authors.

Marra MA et al. (1991) International C. elegans Meeting "CHARACTERIZATION OF THE COSMID C11F2. I. FRAGMENT 'Q' CONTAINS Na+/ H+ ANTIPORTER HOMOLOGOUS SEQUENCES."

Baillie DL, Prasad SS, Marra MA, Clark DV

[International C. elegans Meeting, 1991]

We are involved in a molecular and genetic characterization of that region of the C. elegans genome between /et-56(1V) and unc-22(1V). This 60-100 kb region appears to encode at least four membrane spanning proteins (Fields et al., WBG Vol.11 No.4). We are currently concentrating our analysis on a cosmid that has previously been shown to rescue two alleles of let-56. This cosmid, C11 F2, contains four Pst1 fragments. One of these, an 18 kb fragment known as 'Q', detects a 2.4 kb message on Northern blots. The use of Q as a probe to screen Barbara Meyers' lambda gt10 cDNA library resulted in the isolation of a 1.3 kb cDNA. Sequence analysis of this 1.3 kb cDNA reveals a single long open reading frame of approximately 1200 nucleotides. The inferred amino acid sequence of this open reading frame shows significant similarity (36% amino acid identity) to the 3' portion of the human growth factor activatable Na + / H + antiporter, a protein that makes multiple passes through cell membranes. These transmembrane domains appear to exhibit a high degree of conservation between the predicted C. elegans and human amino acid sequences. The use of this 1. 3 kb cDNA as a probe to screen B. Barstead's lambda ZAP cDNA library has resulted in the isolation of six cDNAs, none of which contain the 5' end of the potential C. elegans Na + / H + antiporter. There appears to be at least two other open reading frames contained within fragment Q, in addition to the antiporter. Sequence analysis of the ends of Q reveal a 270 nucleotide open reading frame progressing into Q from one end, and a similarly sized open reading frame reading out of Q on the opposite end of the fragment. Our efforts are currently directed towards obtaining a full length cDNA, as well as obtaining genomic sequence corresponding to the 5' end of the antiporter. This work has been supported by an NSERC predoctoral scholarship to M.A.M. and an NSERC grant to D.L.B.

Ward S et al. (2000) West Coast Worm Meeting "The promise and peril of genomics: sperm development as model system"

Ward S, Smith HE, Millhouse M

[West Coast Worm Meeting, 2000]

Microarray screening allows the investigator to profile expression patterns on a genome-wide scale. However, even a successful screen can generate a daunting amount of data. In our effort to identify genes involved in sperm development, we compared expression levels in fem-3(gf) mutants, which make only sperm, to fem-1(lf) mutants, which make only oocytes. We identified 650 sperm-enriched genes (as well as 258 oocyte-enriched genes) out of 11,917 genes screened (see Reinke et al.). Now, how do we use this information? We have focused our efforts in two areas: 1) obtaining deletion mutations in potentially interesting genes; and 2) identifying sperm promoter elements and their relevant transcription factors. Prior work with the calmodulin inhibitor trifluoperazine (TFP) had implicated Ca++ function in both sperm activation and motility; therefore, we generated a deletion allele of the sperm-enriched Ca++ channel gene K01A11.4 (since named spe-39). The spe-39 mutant exhibits reduced fertility and an aberrant sperm morphology that mimics TFP treatment. We can now examine the role of other spe mutants in Ca++ sensing, sperm activation, and motility. We are using an in silico approach to identify potential sperm promoter elements, and molecular and genetic analyses to confirm functional significance. The algorithm (written by John Anderson, NCBI) determines which sequences are over-represented in the 5' upstream sequences of sperm genes compared to non-sperm genes. One of these sperm-enriched sequences contains two potential binding sites for the GATA transcription factor elt-1. Prior work has shown that elt-1 is required to specify hypodermal cell fates in the developing embryo; however, our microarray data also identified elt-1 as a sperm-enriched gene. Yeast one-hybrid screening demonstrates that elt-1 binds to and activates transcription from these putative promoter elements. In collaboration with Barbara Page, we are currently investigating the role of elt-1 in sperm development.

Greg J Hermann (2003) International Worm Meeting "Investigating C. elegans apoptosis in an undergraduate Cell Biology course."

Greg J Hermann

[International Worm Meeting, 2003]

For the past two years I have taught an upper division Cell Biology course that utilizes the study of C. elegans apoptosis as the focus of the associated lab. Students in the course have been investigating the staining pattern of a novel monoclonal antibody, F2-P3E3, that was initially isolated due to an intriguing staining pattern in C. elegans embryos that hinted that it might be staining apoptotic cells. While the specific experiments change each year reflecting the progress made on the project the lab is organized to prepare students to be able to design and implement independent projects at the end or the semester, and in some cases beyond. The first month is spent characterizing the antibody staining pattern and C. elegans apoptosis, allowing the students to learn key experimental techniques and become exposed to the research literature introducing them to the significance of their work. The second month is spent answering a question, which for the last two years has been, "Does F2-P3E3 stain cells that are undergoing cell suicide?" In the final month, the students design independent projects that further characterize spatial or temporal aspects of antibody staining, or use the antibody as a novel tool to investigate apoptosis. Prior to the independent projects, proposals are written and anonymously peer reviewed in student run study sections. Following the completion of their independent projects, the lab culminates in an end of the year symposium where the students present their findings and celebrate their accomplishments. In addition to skills gained in designing experiments, reading the primary literature, problem solving, data analysis, and interpretation, students have made remarkable progress in their characterization of the monoclonal antibody. They have shown that (1) apoptosis is necessary for F2-P3E3 staining, (2) F2-P3E3 stains dying cells late in the apoptotic program, likely the engulfed corpse, (3) F2-P3E3 does not stain nuclei, mitochondria, or the microtubule cytoskeleton, and (4) the F2-P3E3 epitope is conserved in other nematode species. This valuable teaching and research resource would not have been identified without the help of Ben Leung and Jim Priess. I must also acknowledge the generosity of members of the C. elegans apoptosis community, in particular Barbara Conradt, for strains, antibodies and advice.

Messom AL et al. (1995) International C. elegans Meeting "A TC1 INSERTION IN PES-1 AND A NEW FORK HEAD HOMOLOGUE."

Messom AL, Hope IA

[International C. elegans Meeting, 1995]

PES-1 [pattern expression site] is a putative transcription factor expressed in the early C. elegans embryo that shows homology to the fork head family of DNA binding proteins [FDPs]. Mutagenesis with the transposable element Tc1 is being used to generate a mutant phenotype for the pes-1 gene. PES-1 is a highly diverged member of the fork head family and it is proposed that it defines a new subgroup within this family. A combination of PCR based and hybridisation screens are being used, to identify homologues of pes-1, to investigate this possibility. We have received the strain NL717 from Ronald Plasterk's group, which contains a Tc1 insertion in the large intron of the pes-1 gene. A screen is currently in progress to identify deletions in the pes-1 gene, arising from excision of the Tc1 element. Through this method we should obtain knockouts of the pes-1 gene, and therefore identify any mutant phenotype, and subsequently be able to further analyse gene function. PES-1 shows only 44% amino acid identity in the putative DNA binding domain with other members of the fork head family. This divergence cannot be explained by it being a C. elegans gene, since two other FDPs have been identified in C. elegans; lin-31 which specifies three alternative vulval fates, identified by Leilani Miller and Stuart Kim; and cfh-1 identified by Marie Azzaria, Barbara Goszczynski and Jim McGhee - both show good homology to the other FDPs. Since pes-1 shows such reduced homology and does not belong to any established subgroup of FDPs, we propose that it may be the founding member of a new subgroup within the fork head family. Degenerate primers were designed to highly conserved regions of the fork head domain focusing on amino acid residues believed to be diagnostic of PES-1. These primers were used in a PCR screen to identify homologues of pes-1, initially using C. elegans genomic DNA. A number of positive results were obtained which are currently being analysed. A new C. elegans gene showing homology to the fork head family has been identified using this approach and is now being characterised.

Kim TY et al. (1996) West Coast Worm Meeting "A NON-NEURONAL SNAP-25-LIKE HOMOLOG IN C. ELEGANS?"

Lee JH, Kim TY

[West Coast Worm Meeting, 1996]

Vesicle fusion complexes are composed of cytosolic proteins such as NSF and SNAP, vesicle membrane proteins such as synaptobrevin, and target membrane proteins such as syntaxin and SNAP-25. Two of the membrane proteins, synaptobrevin and syntaxin, are known to be members of families of genes. SNAP-25, on the other hand, has not been shown to be a gene of a gene family. In yeast, there is only one SNAP-25, called Sec9, which acts at the Golgi to plasma membrane transport step. In higher organisms, only one SNAP-25 homolog has been identified, which acts at the nerve terminal. We wanted to know whether there are other homologs of SNAP-25 that act at other steps of transport and/or in other cells than neural cells. The C. elegans genome project has identified a cosmid (K02D10) that contains a genomic sequence similar to SNAP-25. According to the database, another putative transcript (similar to 4-nitrophenylphosphatase) lies upstream of the SNAP-25-like transcript in the same cosmid. To determine whether these two sequences make a single transcript, we performed a northern analysis with a probe made from the SNAP-25-like sequence. We found that the putative SNAP-25-like sequence makes a separate transcript about 1.1 kb long. We screened a cDNA library and isolated a few clones, none of which contained sequences of the upstream transcript, confirming that the two sequences are not a single gene. We do not know whether these two sequences are polycistronic. We were curious to know the expression pattern of this gene. We made antibodies in rabbits against the gene product and performed whole mount staining using the antibody. The antibody did not stain the nerve cords and nerve cells, which are stained with the antobodies against the genuine SNAP-25. It instead stains specific regions of the cuticle in early larvae. We think that this specific region is the alae of the L1 larvae. I. Hope's LacZ fusion expression data seem to agree with our antibody staining result (personal comm.; also from his home page at the C. elegans www server at UTSW). We plan to identify mutations to determine the defects associated with the disruption of this SNAP-25-like gene. We would like to call this gene nns-1 (Non-Neuronal Snap-25-like ). This work was initiated in Dr. Barbara Meyer's lab when J. Lee was a postdoctoral fellow in her lab.

Bloom L et al. (1991) International C. elegans Meeting "MOLECULAR BIOLOGY OF THE AXON OUTGROWTH GENE unc-76"

Bloom L, Horvitz HR

[International C. elegans Meeting, 1991]

We are interested in understanding the molecular mechanisms by which axons reach their targets. Mutations in the gene unc-76 cause the axons of many neurons to stop prematurely or to take aberrant paths. Most affected axons show defects in anterior or posterior growth along the ventral nerve cord but not in dorsal or ventral growth along the lateral hypodermis. Axon outgrowth of one pair of neurons, the HSNs, is blocked in unc-76 mutant animals when the axons grow in the ventral cord but not when they grow in an alternative lateral position (Steve McIntire, personal communication), suggesting that this gene is involved in the interaction of the axons with their environment rather than in the growth of the axons per se. We have cloned the unc-76 gene by germline transformation with cosmids that map near a restriction fragment length polymorphism associated with the deficiency yDfll (Bob Klein and Barbara Meyer, personal communication) A lO.S kb subclone of cosmid C56C4 completely rescues the Unc phenotype of unc-76(e911) animals, and subclones as small as 5.5 kb rescue the Unc phenotype in young larvae but give only weak rescue in older animals. Using these subclones as probes, we have identified a 1.8 kb transcript present in poly-A(+) RNA from wild-type embryos. We believe that this RNA represents the unc-76 transcript because it is greatly reduced in abundance in unc-76(e911) embryos. We have also used an 8.5 kb subclone to isolate two cDNA clones, 1.8 and 2.6 kb in length, from Stuart Kim's mixed-stage library. These clones contain open reading frames of 422 and 413 amino acids, respectively, and are identical for their first 391 amino acids. Neither contains the 5' end of the gene. A search of the Genbank database revealed no significant similarity to known proteins. The axon guidance defects in unc-76 mutant animals suggest that the gene might act in the neurons affected by unc-76 mutations, in the cells along which their growing axons navigate (other neurons or hypodermis), or in both. To distinguish among these possibilities, we have begun to study the expression of an unc-76-1acZ fusion. We are also examining how expression of the unc-76-1acZ fusion is affected by other mutations that cause axon outgrowth defects similar to those seen in unc-76 mutant animals.