MOLIN Laurent (2003) International Worm Meeting "Study of the CAF1/CCR4/NOT complex in Caenorhabditis elegans"

MOLIN Laurent

[International Worm Meeting, 2003]

In yeast, the CAF1/CCR4/NOT complex, composed of at least eight proteic subunits, has both transcriptional and deadenylase (shortening of the poly(A) tail of mRNAs) activities. Orthologues of most of the subunits of this complex are present in C. elegans and we are interested to study the function of C. elegans CAF1 and other members of this complex. Animals (P0) and their progeny (F1) in which the C. elegans CAF1 homologue, ccf-1, is inactivated by RNAi display several phenotypes :(1) P0 injected or fed with ccf-1 dsRNA are partially sterile, (2) developing F1 embryos display several defects during the first successive divisions and most embryos arrest before reaching the 50 cell stage, (3) a large proportion of F1 animals that reach adulthood are sterile because of arrest of their germ cells at the pachytene stage of the first meiotic division. In F1 males germ cells also arrest in pachytene. Decrease of GLD-1 staining in the most proximal pachytene arrested germ cells nonetheless suggest that these cells are exiting pachytene. Sterile animals often display extrusion of tissue by the vulva, which might result from an abnormal specification of vulval cells. As an other member of the C.elegans CAF1/CCR4/NOT complex is involved in the specification of vulval cells, such a function for ccf-1 is also being investigated. By northern blot, ccf-1 expression is maximal in the germ line and in early embryos, expression is very weak but detectable during other stages of development. Expression is much higher in the hermaphrodite germline than in the male one. Somatic expression is detected in animals without germline. An anti CCF-1 antibody has been produced and is currently being tested. Animals in which the C. elegans orthologue of CCR4 has been inactivated by RNAi do not display any obvious phenotype. By Northern blot, developmental expression of this gene is similar to that of ccf-1, but expression level is similar in hermaphrodite and male germline. Deadenylase activity of both CCF-1 and the orthologue of CCR4 is also being investigated, in vitro by biochemical assays and in vivo by comparing poly(A) tail length of candidate target mRNAs in wild type and sterile worms. CCF-1, by its putative deadenylase activity, could control mRNA translation of genes necessary for pachytene exit, germ cell differentiation and early embryonic development. Post transcriptional repression has been observed for several genes expressed in the germline and, at least for tra-2 and fem-3, such a control is observed in parallel with poly(A) tail length shortening.

Laurent Molin et al. (2002) European Worm Meeting "ccf-1, a putative deadenylase encoding gene, is necessary for meiotic progression."

Laurent Molin, Jean Pierre Magaud

[European Worm Meeting, 2002]

The BTG genes encode a family of proteins with two domains conserved between members and also during evolution. An antiproliferative function has been demonstrated for three members of this family. The unique C. elegans homologue of the BTG genes is fog-3, involved in the differentiation of germ cells into sperm. Yeast two hybrid analysis identified the protein CAF1 (CCR4 associated factor 1) as a protein interacting with BTG1 and 2. CAF1, initially characterized in yeast, is associated to CCR4 in a large transcriptional complex with deadenylase activity.

Laurent Molin et al. (2001) International C. elegans Meeting "Use of C. elegans to study tumour suppressor genes"

Jean Pierre Magaud, Laurent Molin

[International C. elegans Meeting, 2001]

We are studying tumour suppressor genes in normal and neoplastic cells. These genes, whose function is altered in a large number of tumours, have a fundamental role in the cycle cycle, in cellular differentiation, cellular aging or in the response to genotoxic agents. We are using C. elegans in order to better understand the function of these genes and their position within complex signalling pathways. We will more particularly study homologues in C. elegans of the genes BTG-1, BTG-2, FRG-1, FVT-1 and of known interacting proteins. The C. elegans homologue of BTG-1 and BTG-2 is fog-3. Preliminary results will be presented and discussed.

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.

Mergoud dit Lamarche, Adeline et al. (2015) International Worm Meeting "Toward the identification of genetic pathways involved in muscle aging in Caenorahbditis elegans."

Bessereau, Jean-Louis, Gieseler, Kathrin, Solari, Florence, Mergoud dit Lamarche, Adeline, Molin, Laurent

[International Worm Meeting, 2015]

Aging is accompanied by a progressive loss of muscle mass and function, associated with a loss of muscle quality, termed sarcopenia. In human, sarcopenia is responsible for a decrease in mobility leading to a reduction in the quality of life. Epidemiological studies further suggest that skeletal muscle aging is also a risk factor for the development of several age-related diseases such as diabetes, cancer, Alzheimer's disease, and Parkinson's disease.Several mechanisms have been proposed to be involved in muscle aging including mitochondria default [1], apoptosis [2] and alteration of muscle protein turnover [3]. However the physiological importance of these events in the etiology of sarcopenia remains to be investigated.We are using C. elegans to identify genetic pathways involved in muscle aging. For this purpose we first aimed to identify and characterize biomarkers of muscle aging. Previous studies have shown that worms exhibit loss of mobility [4], myosin filament disorganization and change in muscle nuclei shape with age [5]. More recently, the Xu laboratory reported a functional decline of motor neurons that precedes alteration of muscle contraction [6]. However those studies have not allowed identifying genuine muscle biomarker of sarcopenia.We have defined a time course of events that take place during muscle aging starting from a decrease in the expression of some but not all, muscle genes expression, followed by a change in mitochondria morphology and an impairment of muscular proteostasis.Based on these biomarkers, we have generated transgenic lines, thanks to the CRISPR technics, which express fluorescent reporter that mirror the levels of endogenous muscle gene expression. We are now using those strains to perform a screen in order to identify genes that modulate muscle aging in an extensive manner.[1] Romanello V, et al. EMBO J (2010), 29:1774-1785. [2] Marzetti, E., et al. Gerontology, (2012) , 58:99-106 [3] Masiero E, et al. Autophagy ; (2010) 6:307-9. [4] Huang, C., et al. Proc. Natl. Acad. Sci., (2004). 101, 8084-8089. [5] Herndon, et al. Nature, (2002). 419, 808-814 [6] Liu J, et al. Cell Metab (2013) 18(3):392-402.

Saikat Mukhopadhyay et al. (2004) East Coast Worm Meeting "Identification of genes regulating chemosensory neuron-specific morphologies"

Anne Lanjuin, Saikat Mukhopadhyay, Piali Sengupta

[East Coast Worm Meeting, 2004]

Amphid chemosensory neurons sense the external environment via their non-motile cilia, which are specialized for the particular neuron type. For example, AWB has characteristic forked cilia, while AWC has fan-like cilia. How are these structures specified and maintained? FKH-2, a forkhead domain transcription factor is expressed widely in the embryo (Molin et al., 2000) but expression is restricted to the AWB, ASK and ASI amphid neurons in larval stages and adults. In fkh-2 mutants, the ciliary structures of the AWB and AWC neurons but not other neuron types are affected. Dendritic development of AWB and AWC neurons is also affected in fkh-2 mutants, and this effect is manifested as stunting of the dendritic processes as early as the first larval stages. FKH-2 expression in the amphid neurons in early larval stages is regulated by DAF-19, an RFX-type transcription factor regulating general cilia development in all sensory neurons. However, the expression of fkh-2 is partly restored in daf-19 dauers and adults. It is possible that daf-19 could be regulating fkh-2 to determine cell-specific cilia morphology in AWB in addition to its general function in cilia formation. FKH-2 also acts in parallel with the OTX family homeodomain protein CEH-37 to specify the subtype identities of the AWB and ADF sensory neurons. Currently we are screening for additional mutants exhibiting altered cilia morphology specifically in the AWB neurons in order to characterize cell-specific factors necessary for their specialized cilia structure. We are also investigating the role of neuronal activity in the maintenance of ciliary structure. References: Molin L., Mounsey. A., Aslam, S., Bauer, P., Young, J., James, M., Sharma-Oates, A., Hope, I. (2000) Development 127, 4825-4835.

R E Isaac et al. (2005) International Worm Meeting "Structure and function of Caenorhabditis elegans APP-1, an aminopeptidase that is highly expressed in the intestine and in the female germ line"

MR Parsons, DR Brooks, Aj Turner, KAP Payne, R E Isaac

[International Worm Meeting, 2005]

Aminopeptidase P (EC 3.4.11.9, AP-P) is a metallopeptidase that removes amino acids from the N-terminus of peptides with a penultimate N-terminal proline residue. In mammals, a membrane-bound form of AP-P is involved in the regulation of blood pressure through the degradation of the potent vasodilator bradykinin. Hypertension is commonly treated with ACE inhibitors, however potentially fatal side-effects have been reported in individuals with low level serum AP-P activity, thus making the molecular and functional characterization of AP-P of great interest. To improve our understanding of the structure and biological roles of this peptidase, we are currently determining the high-resolution molecular structure and biological function of the C. elegans homologue of human AP-P. The genome of C. elegans contains a single gene (app-1) coding for a cytosolic aminopeptidase P. Recombinant APP-1 containing a N-terminal hexa His-tag was over expressed in E. coli and purified by nickel affinity chromatography. A polyclonal antibody was raised against the recombinant protein and was shown to be specific for APP-1 on western blots. Immunocytochemistry confirmed the intestinal expression of APP-1 previously seen in a GFP promoter-reporter construct produced by Laurent et al. (2001). Interestingly, APP-1 was also shown to be localized in the female germline and in embryos, suggesting roles in oogenesis and embryogenesis. Analytical ultracentrifugation showed that recombinant APP-1 was organized as a dimer. APP-1 was crystallized in space group P212121 with orthorhombic unit cell dimensions 93.7 , 108.25 and 131.28 , suggesting that two protein molecules are present in the asymmetric unit. A full x-ray diffraction data set was collected to a resolution of 2.2 . Laurent, V., D. R. Brooks, et al. (2001). "Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans." European Journal of Biochemistry 268 (20): 5430-5438.

MOLIN, L. et al. (2019) International Worm Meeting "Screen for new actors of muscle aging."

Bessereau, JL., ROY, C., SOLARI, F., MOLIN, L.

[International Worm Meeting, 2019]

We previously demonstrated that muscle aging in C. elegans involves a stereotyped sequence of events characterized by a dramatic decrease in the expression of genes encoding proteins required for muscle contraction, followed by a change in mitochondria morphology, and an increase in autophagosome number (Mergoud et al., 2018). To further characterize the mechanisms underlying muscle aging we used CRISPR/CAS9 to generate a transcriptional reporter that mirrors the down-regulation of tnt-2 (troponine T) expression with age. We used CRISPR-CAS9 to generate a knock-in strain, in which tagRFP-T fluorescence in body wall muscles (BWM) and protein level decrease with age. Using this reporter strain we performed a semi-clonal screen after EMS mutagenesis in order to identify genes whose mutation maintains tagRFP-T fluorescence with age. Details of the screening strategy and some preliminary results will be presented. Mergoud dit Lamarche A., Molin L., Pierson L., Mariol MC., Bessereau JL., Gieseler K., Solari F. (2018). UNC-120/SRF independently controls muscle aging and lifespan in Caenorhabditis elegans. Aging Cell. https://doi.org/10.1111/acel.12713

Payne KAP et al. (2004) European Worm Meeting "STRUCTURE AND FUNCTION OF AMINOPEPTIDASE P FROM CAENORHABDITIS ELEGANS"

Isaac RE, Brooks DR, Parsons MR, Turner TJ, Payne KAP

[European Worm Meeting, 2004]

Aminopeptidase P (EC 3.4.11.9, AP-P) is a metallopeptidase that removes amino acids from the N-terminus of peptides with a penultimate N-terminal proline residue. In mammals, a membrane-bound form of AP-P is involved in the regulation of blood pressure through the degradation of the potent vasodilator bradykinin. Hypertension is commonly treated with ACE inhibitors, however potentially fatal side-effects have been reported in individuals with low level serum AP-P activity, thus making the molecular and functional characterization of AP-P of great interest. To improve our understanding of the structure and biological roles of this peptidase, we are currently determining the high-resolution molecular structure and biological function of the C. elegans homologue of human AP-P. The genome of Caenorhabditis elegans contains a single gene (app-1) coding for a cytosolic aminopeptidase P. Recombinant APP-1 containing a N-terminal hexa His-tag was over expressed in E. coli and purified by nickel affinity chromatography. A polyclonal antibody was raised against the recombinant protein and was shown to be specific for APP-1 on western blots. Immunocytochemistry confirmed the intestinal expression of APP-1 previously seen in a GFP promoter-reporter construct produced by Laurent et al (2001). Interestingly, APP-1 was also shown to be localized in the female germline and in embryos, suggesting roles in oogenesis and embryogenesis. Analytical Ultracentrifugation was used to show that recombinant APP-1 was mainly organized as a dimer. Recently APP-1 was crystallized in space group P212121 with orthorhombic unit cell dimensions 93.7 , 108.25 and 131.28 , suggesting that two protein molecules are present in the asymmetric unit. A full x-ray diffraction data set was collected to a resolution of 2.2 . Laurent, V., D. R. Brooks, et al. (2001). Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans. European Journal of Biochemistry 268(20): 5430-5438.

Gross, E. et al. (2013) International Worm Meeting "The neuroglobin GLB-5 regulates C. elegans responses to hypoxic exposure."

Soltesz, Z., Gross, E., Zelmanovich, V., de-Bono, M.

[International Worm Meeting, 2013]

Fine-tuned O2 metabolism is essential for most animals, requiring animals to monitor and adapt to changes in O2 levels. We previously identified a polymorphic neuroglobin, GLB-5, that acts in O2-sensing neurons and enables C. elegans to respond to small changes in O2 concentration (Persson et al, 2009). Here we show that GLB-5 is essential for fast behavioural recovery after exposure to hypoxia. Whereas glb-5(Haw) animals recovered from hypoxia within minutes, glb-5(Bri) recovered slowly, over four hours. By combining genetics, biochemistry, and Calcium imaging we provide evidence that glb-5 enables fast recovery of O2-sensing neurons after prolonged hypoxic exposure. We designed mutagenesis screens to explore how GLB-5 regulates recovery from hypoxia. One mutant we identified disrupts a conserved chaperone that regulates the activity of the O2 sensing neurons. The chaperone regulates the spatial organization of soluble guanylate cyclases in these neurons, altering the way they interact with the neuroglobin to control the O2 response properties of these neurons. Persson A, Gross E, Laurent P, Busch KE, Bretes H, de Bono M (2009) Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans. Nature 458: 1030-1033.