Ingrid Masse et al. (2005) International Worm Meeting "Lifespan and dauer regulation by tissue-specific activities of Caenorhabditis elegans DAF-18."

Marc Billaud, INGRID MASSE, Florence Solari, Laurent Molin

[International Worm Meeting, 2005]

In Caenorhabditis elegans, the insulin/IGF1 DAF-2 receptor controls entry into dauer and longevity. DAF-2 signalling cascade includes the PI3 Kinase homolog AGE-1 and the FOXO transcription factor DAF-16. The DAF-2 pathway is downregulated by DAF-18 which is encoded by the ortholog of the human tumor suppressor gene PTEN. We have previously shown that, like PTEN, DAF-18 antagonizes the activity of PI3 kinase/AGE-1. To further explore the role of DAF-18 in the regulation of the insulin pathway, we investigated which tissue(s) DAF-18 functions in to regulate dauer formation and lifespan. Our data show that complete dauer formation requires daf-18 expression in several tissues and that the remodelling of dauer tissues depends on both cell autonomous and cell nonautonomous daf-18 function(s). Conversely, daf-18 expression increases adult lifespan in all individual tissues tested. Furthermore, we show that the role of DAF-18 in dauer and lifespan control depends on DAF-16 activation, which is regulated by both cell autonomous and cell nonautonomous DAF-18 function(s) and in a tissue specific manner. Overall our data strongly suggest that several tissues act as signalling centres to mediate DAF-18 function and that DAF-18 could act outside the canonical DAF-2/DAF-16 pathway to regulate dauer and lifespan.

Ingrid Masse et al. (2006) European Worm Meeting "Identification of a Novel Protein Kinase that Acts in the DAF-18/PTEN Pathway and Regulates Lifespan"

Florence Solari, Marc Billaud, INGRID MASSE, Laurent Molin

[European Worm Meeting, 2006]

Ingrid Masse, Laurent Molin, Marc Billaud and Florence Solari. In Caenorhabditis elegans, the insulin/IGF-1 DAF-2 receptor controls entry into dauer and longevity. DAF-2 activates the PI3 kinase homologue AGE-1 which results in phosphorylation and cytoplasmic sequestration of the DAF-16 transcription factor. Activation of the DAF-2 pathway is downregulated by DAF-18 which is encoded by the orthologue of the PTEN human tumor suppressor gene. We have previously shown that DAF-18 antagonizes the activity of the PI3 kinase/AGE-1 and that this function requires the DAF-18 lipid phosphatase activity. To better characterize the pathways involving DAF-18, we have initiated a RNAi screen aimed at identifying novel genes that regulate lifespan and that genetically interact with daf-18. We focused our initial screening on the C. elegans kinome that comprises 438 genes coding for protein kinases. Using this strategy, we have identified a new interactor of daf-18 that controls lifespan. We found that this kinase acts upstream of the daf-16/FOXO transcription factor but in parallel of the canonical daf-2/age-1 pathway. We further established that worms with an inactivation of this gene were resistant to oxidative stress. More information on the nature and the mode of action of this protein kinase will be presented at the meeting.

Ingrid Masse et al. (2008) European Worm Meeting "A novel role for the SMG-1 kinase in lifespan and oxidative stress resistance in Caenorhabditis elegans."

Laurent Molin, Laurent Mouchiroud, Florence Solari, Philippe Vanhems, Francesca Palladino, Marc Billaud, INGRID MASSE

[European Worm Meeting, 2008]

Type Body t The PTEN tumour suppressor encodes a phosphatase, and its daf-. 18 orthologue in Caenorhabditis elegans negatively regulates the. insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and. other species. In order to identify new DAF-18 regulated pathways involved. in aging, we initiated a candidate RNAi feeding screen for clones that. lengthen lifespan. Here, we report that smg-1 inactivation increases. average lifespan in a daf-18 dependent manner. Genetic analysis is. consistent with SMG-1 acting in parallel to the canonical DAF-2 receptor. pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a. serine-threonine kinase which plays a conserved role in nonsense-mediated. mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also. been implicated in the p53-mediated response to genotoxic stress. The. effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD. function, but requires the p53 tumour suppressor orthologue cep-1.. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in. a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of. SMG-1 in lifespan regulation depends on its function in oxidative stress. resistance. Taken together, our results unveil a novel role for SMG-1 in. lifespan regulation.. ext here. Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.

Dolanchanpa Ghosh et al. (2003) International Worm Meeting "Structure-function analysis of the PIE-1 protein to elucidate its role in germline development."

Dolanchanpa Ghosh, Geraldine Seydoux

[International Worm Meeting, 2003]

C.elegans protein PIE-1 is a component of the germ plasm essential for the specification and development of the embryonic germline. PIE-1 has at least two functions in germline blastomeres: it represses mRNA transcription in the nuclei, and regulates the expression of the maternal RNA, nos-2 in the cytoplasm. We are interested in how these two functions are carried out and how they influence germline development. Our approach is to create transgenic worms that express the PIE-1 protein bearing mutations in different domains. Conventional transformation methods yield transgenes that are quickly silenced in the germline, making detailed phenotypic analysis difficult.We have therefore adopted the microparticle bombardment method to create low-copy integrative transformants(1). Ingrid Dagostino in the lab has created a GATEWAY-based pie-1 expression vector, containing unc-119 gene as a selection marker. Using this vector, Ingrid was able to obtain a GFP:PIE-1 line, which fully rescues a pie-1(0) mutant and has remained stable for over 20 generations. We are now in the process of generating similar lines with mutant versions of PIE-1. 1.Praitis V, Casey E, Collar D,and Austin J.(2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 2001 Mar;157(3):1217-1226

Florence Solari et al. (2003) International Worm Meeting "The human tumor suppressor PTEN regulates longevity and dauer formation in Caenorhabditis elegans"

ANDREW ML CHAN, ANGELIQUE BOURBON, INGRID MASSE, BERNARD PAYRASTRE, Gary Ruvkun, Patrick Hu, Florence Solari, Marc Billaud

[International Worm Meeting, 2003]

The PTEN tumor suppressor is a dual-specificity phosphatase that dephosphorylates phosphatidylinositol 3, 4, 5 triphosphate (PIP3) and protein substrates. PTEN function is modulated by its carboxy terminal region which contained several clustered phosphorylation sites and a PDZ-binding motif. Although PTEN growth suppression effect is well demonstrated, its additional biological roles are less well understood. DAF-18, a Caenorhabditis elegans homolog PTEN, is a component of the insulin/IGF-I signalling pathway that controls entry at the dauer larval stage and adult longevity. To explore in a model organism the ability of human PTEN to regulate the insulin signalling cascade and its possible involvement in the mechanisms of aging, we undertook a study of PTEN function in C.elegans. We now report, that PTEN expression can substitute for DAF-18 and restores the dauer and longevity phenotypes in mutant worms devoided of DAF-18. Furthermore, we provide genetic and biochemical evidence that entry in the dauer stage and lifespan depends on PTEN-mediated regulation of PIP3 levels. Finally, we established that the phosphorylation sites of the PTEN tail and the PDZ-binding motif are necessary for PTEN ability to control the transduction of the insulin/IGF-I signal. These results demonstrate that PTEN negatively regulates the insulin/IGF pathway in C.elegans, thus raising the possibility that PTEN acts similarly in mammals and may be also involved in the aging process.

Philip S Hartman et al. (2005) International Worm Meeting "Chromosome dosage as a life span determinant in C. elegans."

Naoaki Ishii, Philip S Hartman, Terrell Carter

[International Worm Meeting, 2005]

Gender differences in lifespan have been observed repeatedly, with females generally living longer than males. Various explanations have been advanced and are generally based upon phenotypic differences derived from physiological, behavioral or anatomical sexual dimorphisms. C. elegans hermaphrodites live longer than males when cultured en masse. In contrast, males live longer than hermaphrodites when cultured individually. Gems and Riddle convincingly demonstrated that certain male-specific behaviors are detrimental to survival and act to limit the life span of wild type males. However, since hermaphrodites contain a pair of X chromosomes (XX) and males are XO, we questioned whether chromosomal differences per se might also impact lifespan. The use of mutations in the sex-determination genes tra-1 and her-1 allowed us to uncouple sexual phenotype from the normal X chromosomal composition and demonstrate that possession of two X chromosomes limits hermaphrodite life span. We wondered whether diplo-X animals might live shorter than haplo-X animals because faulty dosage compensation results in inappropriately high expression of X-linked genes in geriatric animals. This hypothesis carries two predictions. First, mutants with faulty dosage compensation ought to be short lived. This proved true, as three dosage-compensation-defective Dpy mutants were short lived, but four other Dpy mutants with normal dosage compensation had normal life spans. The second prediction is that age-specific alterations in dosage compensation should be reflected in a global elevation of mRNA levels of X-linked genes relative to the autosomes. We employed the microarray data generated by Lund and coworkers to show that X-linked gene expression in the roughly 10 percent of geriatric worms that were still alive between 16 and 19 days was almost 20 percent higher than autosomal gene expression. In concert, our data suggest that age-related amelioration of dosage compensation occurs in this nematode and, as a consequence, limits the life span of XX animals. As such, this represents the first example in which this hypothesis is supported by both mortality and molecular data.

Rose J et al. (2000) West Coast Worm Meeting "Differential effects of heat shock and cold shock following massed and distributed long-term habituation training in C. elegans"

Rankin CH, Rose J, Eng K

[West Coast Worm Meeting, 2000]

A new en masse long-term habituation (LTH) training procedure was used to examine the effects of heat and cold shock on LTH of the tap withdrawal response. In order to induce LTH a distributed training protocol was used where groups of worms received four blocks of 20 taps separated by one-hour rest periods and testing each worm individually 24 hours later. LTH following massed training was also examined by administering one block of 120 taps to worm groups. Worms that received massed training did not show LTH when tested 24 hours later. Temperature shock was administered by submerging sealed worm-containing petri plates into either a 32 C (heat shock) or a 0 C bath for the first 40 minutes following training blocks: worms were kept at room temperature for the last 20 minutes. Worms in the control condition underwent similar submersion during rest periods at 21 C (room temperature). It was found that when heat shock is administered between distributed training blocks an attenuation of LTH results. Predictably, heat shock after massed training did not produce LTH. Interestingly, when worms receive distributed training blocks with cold shock between blocks, LTH of the tap withdrawal response is at least retained and may be enhanced. Similar to before, LTH did not result from massed training followed by cold shock. Twelve-hour retention has also been examined with distributed training and similar findings resulted where LTH was induced after room-temperature submersion and cold shock but not heat shock. Currently, studies are investigating if massed training results in LTH if measured 12 hours rather than 24 hours later. As well, the effects of heat shock and cold shock on 12-hour retention following massed training are being examined. The results that have been reported thus far indicate that differential mechanisms may underlie both LTH resulting from massed versus distributed training and the effects of heat shock versus cold shock. Research funded by the Natural Sciences and Engineering Research Council of Canada

Jacoby M et al. (1996) East Coast Worm Meeting "Improved gene trap screens"

Jacoby M, Fire A, Macarah C

[East Coast Worm Meeting, 1996]

A genetrap screen provides an efficient means for identification of genes expressed in specific cell types (under specific) physiological conditions. Briefly, a population of random fragments from genomic DNA are inserted upstream of a reporter gene (such as lacZ) in a standard expression vector, to generate a library of potential gene fusions. Clones from the library are then injected (in pools or en masse) and the resulting transformed lines examined for reporter activity pattern. Genetrap methods for C. elegans were pioneered by Ian Hope [1], who identified a variety of differentially expressed genes. Seydoux, Godbey, and Fire [2] used a variant of this protocol to identify a number of genes active in the early embryo. Due to the nature and inefficient expression of the reporter constructs used, previous genetrap screens may have been biased toward detection of highly expressed genes. Recently, a variety of reporter constructs with improved expression have been developed in this lab [3]. The primary feature of these vectors is the presence of multiple synthetic intron sequences interspersed throughout the reporter coding region. Our most sensitive genetrap vectors make use of a lacZ reporter containing 12 introns [3]. A library of approximately 6000 potential gene fusions has been constructed using this reporter. We have developed a set of streamlined protocols for culture, pooled DNA isolation, and for isolation of active constructs from pools. Clones from the library are being injected in pools of 96 to form transgenic lines. To date, 960 clones have been screened and a N ariety of different staining patterns obtained. Three of these patterns are being further analyzed: two give staining in midstage embryos and one gives intrinsic coeloemocyte staining. In addition to the genetrap using a lacZ reporter, we have initiated similar screens using a modified reporter that has both intervening sequences and mutations that improve the fluorescence properties of gfp [4]. These "green fusion" libraries should have the advantage of allowing the fusion products to be localized intracellularly and followed dynamically in live worms. 1. Hope, I. (1991) Development 113, 399 2. Seydoux, G. and Fire, A. (1992) WBG 12#4,p. 20; A. Godbey, pers. comm. 3. Fire, A. and Xu, S. (1994) WBG 1344, p. 29 4. Heim, R., Cubitt, A., and Tsien, R. (1995) Nature 373, 663