ABSTRACT BOOK Vermidi XX ENS Lyon Amphithéâtre Mérieux

ABSTRACT BOOK VerMidi XX ENS Lyon Amphithéâtre Mérieux

2017 VERMIDI XX PROGRAM

9:30 - 10:00 Welcome Breakfast 10:00 - 10:45 Keynote Lecture: Thorsten Hoppe Ubiquitin sets the timer: coordination of aging and proteostasis 10:45 - 12:00 Session 1

10:45 - 11:05 Céline Jenzer (I2BC, Gif sur Yvette) Determination of functional speciﬁcity of autophagic proteins during phagocytosis of apoptotic cells in embryo. 11:05 - 11:25 Philippe Tardy (INMG, Lyon) ß-spectrin and ankyrin regulate K2P channel function and localisation in C. elegans 11:25 - 11:45 Georgia Rapti (Rockefeller University, New York) Glia and pioneer neurons initiate C. elegans brain assembly through non-canonical Chimaerin/Furin axon guidance 11:45 - 12:00 A word from our sponsors 12:00 - 14:30 Lunch / Poster Session

14:30 - 15:50 Session 2

14:30 - 14:50 Germano Cecere (Institut Pasteur, Paris) small RNAs in epigenetic inheritance 14:50 - 15:10 Denis Dupuy (IECB, Bordeaux) Quantitative meta analysis of RNAseq data reveals a stochastic component of alternative splicing and transplicing in C. elegans 15:10 - 15:30 Lise Frézal (IBENS, Paris) Evolutionary variation in the heat-triggered mortal germ line phenotype in C. elegans 15:30 - 15:50 Frances Edwards (IJM, Paris) BUB-1 is at the core of antagonistic activities in the establishment of kinetochore attachments to the mitotic spindle

15:50 - 16:20 Cofee break

16:20 - 17:30 Session 3

16:20 - 16:40 Rodrigo Caceres (Institut Curie, Paris) Role of acto-myosin cytoskeleton in cell invasion in Caenorhabditis elegans 16:40 - 17:00 Xinyi Yang (IBPS, Paris) Rotating and elongating embryos: SPIM microscopy reveals how C. elegans embryos extend through a ratchet mode

17:00 - 17:20 Grégoire Michaux (IGDR, Rennes) Coordinated morphogenesis through tension induced planar polarity

17:30 Happy Hour

Amphitéâtre Charles Mérieux DIRECTIONS

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Determination of functional speciﬁcity of autophagic proteins during phagocytosis of apoptotic cells in embryo., C´eline Jenzer [et al.] ...... 5

ß-spectrin and ankyrin regulate K2P channel function and localisation in C. elegans, Philippe Tardy [et al.] ...... 6

Glia and pioneer neurons initiate C. elegans brain assembly through non-canonical Chi- maerin/Furin axon guidance, Georgia Rapti [et al.] ...... 7

small RNAs in epigenetic inheritance, Germano Cecere ...... 8

Quantitative meta analysis of RNAseq data reveals a stochastic component of alternative splicing and transplicing in C. elegans., Nicolas Tourasse [et al.] ...... 9

Evolutionary variation in the heat-triggered mortal germ line phenotype in C. elegans., Lise Fr´ezal[et al.] ...... 10

BUB-1 is at the core of antagonistic activities in the establishment of kinetochore attachments to the mitotic spindle., Frances Edwards [et al.] ...... 12

Role of acto-myosin cytoskeleton in cell invasion in Caenorhabditis elegans, Rodrigo C´aceres [et al.] ...... 13

Rotating and elongating embryos: SPIM microscopy reveals how C. elegans embryos extend through a ratchet mode, Xinyi Yang [et al.] ...... 14

Coordinated morphogenesis through tension induced planar polarity, Ghislain Gillard [et al.] ...... 15

A spectrin/PAK/actin-capping network stabilizes cell shapes in a morphogenetic ratchet process during C. elegans elongation, Gabriella P´asti [et al.] ...... 16

CRLD-1A is a membrane-associated protein disulﬁde isomerase required for acetylcholine receptor biogenesis, Manuela D’Alessandro [et al.] ...... 17

1 Mitochondria dynamics modulation and WAH-1 (AIF homolog) diminution reduce physiopathological muscle degeneration in C. elegans., Charlotte Scholtes [et al.] ...... 18

EPIDERMAL RESPONSE TO FUNGAL INFECTION AND WOUNDING IN C. EL- EGANS, Clara Ta↵oni [et al.] ...... 20

Exploring fungal virulence using C. elegans, Xing Zhang [et al.] ...... 21

Channel Nucleoporins recruit the Polo-like kinase PLK-1 to Nuclear Pore Complexes in prophase to direct Nuclear Envelope Breakdown, Lisa Martino [et al.] ...... 22

Mechanisms of immune activation after cuticle damage, Jean-Christophe Lone [et al.] .. 23

Fonctional study of Katanin, a prototype of microtubule-severing enzyme essential for C.elegans meiosis, Nicolas Joly [et al.] ...... 25

An Atypical Mechanism of Chromosome Segregation in the C. elegans Oocyte, Kimberley Laband [et al.] ...... 26

Deciphering the role of Notch signaling in Y to PDA transdi↵erentiation in vivo in c.elegans., Laura Vibert [et al.] ...... 27

Actin cytoskeleton in cell invasion and migration in Caenorhabditis elegans vulval development, Nagagireesh Bojanala [et al.] ...... 28

Cellular innovations at the origin of pseudogamy in nematodes, Manon Grosmaire [et al.] 30

Characterization of the SET1/MLL complexes in C.elegans, Flore Beurton [et al.] .... 31

Post-transcriptional histone modiﬁcations maintain cell identity and genome stability in the Caenorhabditis elegans germline, Marion Herbette [et al.] ...... 32

Asymmetric cell division and the cytoskeleton in nematodes, Majdouline Abou-Ghali [et al.] ...... 33

Analysis of syndecan function at the C. elegans neuromuscular junction., Camille Va- chon [et al.] ...... 34

Identiﬁcation of novel regulators of GABAergic synaptogenesis in the nematode Caenorhab- ditis elegans., Marine Gueydan [et al.] ...... 35

The role of sel-10 in transdi↵erentiation, C´ecile Delance [et al.] ...... 36

Analysis of activity-dependent synaptogenesis at the SAB neuromuscular junction., Alexis Weinreb [et al.] ...... 37

2 A Simple and rapid high throughput method to isolate large male populations., Aniela Zablocki [et al.] ...... 38

Combining optogenetic and transcriptomic approaches to dissect thermal nociceptor habituation in C. elegans using LiTeSt: a versatile Light and Temperature Stimulation platform, Andrei-Stefan Lia ...... 39

FRET imaging and thermalized microﬂuidics: a combined approach to monitor the C. elegans CaMK-CREB pathway in vivo, Gabriella Saro [et al.] ...... 40

Screening for nociception genes in C. elegans using an optogenetic approach, Filipe Marques 41

Identiﬁcation of novel regulators of the two-pore domain potassium channel EGL-23 in Caenorhabditis elegans, Sonia El Mouridi [et al.] ...... 42

wrmScarlet, seeing is believing., Sonia El Mouridi [et al.] ...... 44

COULD OUR LIFE BE SWEETER AND LONGER?, Alexia Gomez [et al.] ...... 45

List of participants 45

3 KEYNOTE LECTURE

Ubiquitin sets the timer: coordination of aging and proteostasis

Thorsten HOPPE

University of Cologne Germany.

4 Determination of functional speciﬁcity of autophagic proteins during phagocytosis of apoptotic cells in embryo.

1 1 1 1 C´eline Jenzer ⇤ , Elena Simionato , C´eline Largeau , Renaud Legouis†

1 I2BC – Centre National de la Recherche Scientiﬁque - CNRS – Avenue de La Terrasse, 91198 Gif sur Yvette, France

Phagocytosis and autophagy are two lysosome-mediated processes involved in the clearance of extracellular and intracellular components, respectively. These two processes are involved in various human diseases such as cancers. Previously, the laboratory has shown a sequential and specific involvement of autophagic proteins in the autophagic cascade [1,2]. Recent studies have identified the recruitment of autophagic proteins during phagocytosis of apoptotic corpses in the so called LC3-associated phagocytosis (LAP) [3]. LAP is a distinct process from autophagy but it relies on some members of autophagy pathway to allow an ecient degradation of the phagocytosed cargo. The objective of this study is to elucidate the physiological and pathological roles of autophagy in the phagocytosis of apoptotic corpses in Caenorhabditis elegans. Using time-lapse microscopy, electron microscopy and genetic approaches, we analyzed the function of the LC3 homologues, LGG-1 and LGG-2, during this process. In this study, we showed that these proteins are involved in the phagocytosis of apoptotic cells. Indeed, lgg-1 and lgg-2 mutants present an enhanced number of apoptotic cells compared to wild type embryos. Moreover, di↵erences in LGG-1 and LGG-2 expression profile in apoptototic corpses and phagocytic cells demonstrated di↵erential participation of the two proteins in the clearance of apoptotic corpses. Our results suggest that LGG-1 has a function link to the autophagy in the apoptotic cell whereas LGG-2 is involved in phagocytic cells in later steps of maturation/degradation of the phagosome. Manil-Ségalen, M. et al. The C. elegans LC3 Acts Downstream of GABARAP to Degrade Autophago- somes by Interacting with the HOPS Subunit VPS39. Dev. Cell 28. 43–55 (2014).

Jenzer, C. et al. Human GABARAP can restore autophagosome biogenesis in C. elegans lgg 1 mutant. Autophagy 10. 1868–1872 (2014). Martinez, J. et al. Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the ecient clearance of dead cells. Proc. Natl. Acad. Sci. U. S. A. 108. 17396–17401 (2011].

⇤Speaker †Corresponding author: [email protected]

5 ß-spectrin and ankyrin regulate K2P channel function and localisation in C. elegans

1 2 1 Philippe Tardy ⇤ , Marine Mercier , Thomas Boulin

1 Institut NeuroMyoGène (INMG) – UniversitéClaude Bernard-Lyon I - UCBL (FRANCE) – 8 RUE RAPHAEL DUBOIS, 69100 Villeurbanne, France 2 Centre de génétique et de physiologie moléculaire et cellulaire (CGphiMC) – CNRS : UMR5534, Université Claude Bernard - Lyon I (UCBL) – UNIVERSITE CLAUDE BERNARD LYON 1 Bâtiment Gregor Mendel 16 rue RaphaëlDubois 69622 VILLEURBANNE CEDEX, France

Two-pore domain potassium channels (K2P) compose a family of highly conserved potassium- selective ion channels that establish and maintain the electrical membrane potential. Despite their fundamental role, comparatively little is known about the cellular processes that control the number, the activity and the subcellular localization of K2P channels in vivo. We combine genetic screening with state-of-the-art genome engineering to address this question. Mutants of the UNC-58 K2P channel have strong phenotypes in C. elegans. Loss-of-function mutants show signiﬁcantly decreased mobility, comparable to the loss of the main cholinergic receptor at neuromuscular junctions. This suggests an important role for UNC-58 in the neuromuscular system. Consistently, we observe an expression of the channel in skeletal muscles and in cholinergic motor neurons, using knockin strains obtained by CRISPR/Cas9 genome engineering.

Gain-of-function mutants are unable to roam freely around the plate and display a unique ﬂipping behavior, whereby worms rotate around their antero-posterior body axis. In a genetic suppressor screen for worms that regain a more wild-type mobility, we identiﬁed a mutant in unc44 /ankyrin.Inthis mutant, unc-58 is no longer localized at the membrane, but relocated to an intracellular compartment. Interestingly, the mutation is a point mutation close to the binding site of ankyrin to Phosphatidyli- nositol (3,4,5)trisphosphate (PIP3).In previous studies, ankyrin binding to PIP3 has been shown to be required for actin-dependent cargo transport.

We therefore hypothesize that ankyrin is implicated in the proper transport of the UNC-58 K2P channel to membrane surface.

We also identiﬁed three alleles of unc-70 /ß-spectrin, a well-known physical interactant of ankyrin. Surprisingly in these mutants, UNC-58 is still localized at the muscle surface, which suggests that two parallel pathways control the function (ßspectrin) and the localization (ankyrin) of the UNC-58 channel in C. elegans muscle.

⇤Speaker

6 Glia and pioneer neurons initiate C. elegans brain assembly through non-canonical Chimaerin/Furin axon guidance

1 1 Georgia Rapti ⇤ ,ShaiShaham

1 The Rockefeller University, New York (RU) – 1230 York avenue, The Rockefeller University, New York, Shaham Lab, United States

Central nervous system (CNS) assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells to form tracts guiding follower axons. Yet, identities of pioneer neurons and guidance substrates, and their interactions, are unclear. Glia can regulate axon navigation, yet detailed molecular mechanisms governing this activity are not well understood. To determine how neurons and glia cooperate for CNS formation, we are studying assembly of the C. elegans nerve ring (NR), the CNS-like neuropil, which consists of ˜180 axons enveloped by four astrocyte-like CEPsh glia. Using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans NR assembly. CEPsh glia are key for assembly initiation, regulating both pioneer and follower axon guidance. We identify pioneer neurons, with unique growth properties, anatomy and innervation, that cooperate with glia to guide follower axons of diverse groups, resulting in orderly assembly of the NR. We identify six known guidance cues controlling NR assembly, and demonstrate that CEPsh glia guide pioneer and follower axons through distinct guidance cues.

We isolated a new mutant that a↵ects axon guidance and not outgrowth initiation. A GTPase regulator and a pro-hormone convertase, both defective in this mutant, regulate guidance-cue tracking, and function in glia for pioneer-neuron guidance, and in both glia and pioneer-neurons for follower axon navigation. Moreover, while this double mutant blocks 70% of NR axon entry, each lesion alone results in only mild NR defects (5-15%). We used the genetic bottleneck revealed by the double mutant to study redundancies in CNS formation, a problem that has plagued genetic analysis of the process. In screens for novel redundant axon-guidance genes, we isolated over 20 mutants, several of which appear to be in previously unknown genes, based on whole-genome sequencing. Taken together, our studies suggest a pivotal role for glia in initiation of CNS assembly and open the door to uncovering previously unrecognized axon guidance genes. Embryonic CEPsh glia are reminis- cent of vertebrate radial glia, whose molecular biology is largely uncharacterized. Furthermore, the mammalian homologs of the genes we identify have known axon guidance roles in vertebrates. Our studies, therefore, may reveal conserved mechanisms promoting CNS assembly.

⇤Speaker

7 small RNAs in epigenetic inheritance

1 Germano Cecere ⇤

1 Institut Pasteur (IP) – Institut Pasteur – 25 Rue du Docteur Roux, France

Epigenetic mechanisms are considered to be central to the development of multicellular organisms made of di↵erent cell types, all having identical genomes. Yet, the molecular mechanisms regulating how epigenetic traits can be inherited during cell division or across generations are not fully understood. The nematode Caenorhabditis elegans is an excellent animal model system to study epigenetic inheritance because of its short life cycle and ability to grow large isogenic populations of worms. Moreover, transgenerational epigenetic phenomena have been already described in this system. We are focusing our research in characterizing the role of small RNAs in transmitting epigenetic traits across generations. We are currently working on speciﬁc classes of small RNAs that are produced in the germline of the animal and possibly inherited across generations. We aim to characterize (i) their ability to propagate the memory of active transcription during early embryonic development, and (ii) to test their role in propagating the memory of stress responses across generations to facilitate the adaptation of animals to environmental changes. We are integrating genetic, biochemical, and molecular biology tools with high-throughput genomic and proteomic approaches to achieve a deep mechanistic understanding of the role of small RNAs in epigenetic inheritance in animals.

⇤Speaker

8 Quantitative meta analysis of RNAseq data reveals a stochastic component of alternative splicing and transplicing in C. elegans.

1 1 1 Nicolas Tourasse , Jonathan Millet , Denis Dupuy ⇤

1 Laboratoire ARNA, Inserm U1212, CNRS UMR5320 , Institut Europ´eende Chimie et Biologie (IECB) – Inserm : U1212 – 2, rue Robert Escarpit, 33607 Pessac, France, France

Alternative splicing, allows a single gene to encode several protein variants, called isoforms, with altered stability, localization, specificity or activity. Despite the accumulation of transcriptomics data over the past decade it remains unclear what proportion of alternative splicing is actively regulated and what fraction reflects imperfect processing by the spliceosome. Here we used a compendium of ˜2000 C. elegans RNAseq datasets to expand the dynamic range of detection of RNA isoforms and discriminate low abundance RNA species based on the reproducibility of their detection. This allowed to us to detect a large number of previously undetected exon-exon junctions and previously unreported transplicing sites (potentially indicative of alternative promoter usage). For each alternatively splice junction we could compute a ”usage frequency” representing the relative inclusion of each exonic fragment. We found that a significant fraction of predicted alternative splicing events only participate to a very low proportion of the transcripts from their respective genes. These rarely used splice sites are signifi- cantly less conserved in other nematode genomes than splice sites with a higher usage frequency. Similarly, minor transplicing sites seem to indicate a base line of sloppiness in the Splice Leader attachment mechanism accounting for a large fraction of the detected events. Still our analysis detected about 85% of genes being actively transpliced and seem to indicate that the remaining 15% are missed due to sensitivity limitations even at ur level of coverage. We propose to add quantitative expression information on the Wormbase gene model to allow users to visualize at a glance the relative expression of each detected isoforms

⇤Speaker

9 Evolutionary variation in the heat-triggered mortal germ line phenotype in C. elegans.

1 1 Lise Fr´ezal ⇤ , Gaotian Zhang , Miska Eric† , Marie-Anne F´elix‡

1 Institut de biologie de l’école normale supérieure – Ecole´ normale supérieure [ENS] - Paris, Inserm : U1024, CNRS : UMR8197 – Paris, France

The mortal germ line phenotype was defined by Ahmed and Hodgkin as a multigenerational phenotype, whereby a selfing C. elegans line becomes sterile after several generations (1). The C. elegans N2 reference strain does not display this phenotype in laboratory conditions, but Ahmed and Hodgkin could isolate mutants that are mortal. Some of these mutations are temperature-sensitive and a↵ect small RNA and chromatin modification pathways, which may explain the multigenerational nature of the phenotype (e.g. 2,3). We discovered serendipitously that many C. elegans wild isolates display a strong mortal Germ Line (Mrt) phenotype (1) at high temperature, whereby upon chronic exposure to temperatures such as 25 C, they progressively become sterile after several generations. We report here on this surprising finding.

We ﬁrst assayed a reference panel of 97 genetically distinct isogenic C. elegans wild isolates (4) for the number of generations at 25 C after which full sterility occurred. Reproducible variation was found among these wild isolates. For example, some isolates, such as MY10, become sterile after 3 generations at 25 C, while others, such as JU397 become sterile after 7-10 generations. We found no correlation between the severity of the Mrt phenotype and the climatic conditions of C. elegans isolation.

We observed a progressive brood size decrease and germ line defects in the generations before full sterility. The mortal germline phenotype was however fully reversed by switching back from 25 Cto 15 C before the last generation. Most surprisingly, germline immortality of some isolates was rescued by artiﬁcial infection with Nematocida microsporidia, natural intracellular pathogens of C. elegans that infect the intestinal cells. This rescue by Nematocida infection thus suggests signalling from soma to germline. We suggest that the mortal germ line phenotype observed under laboratory conditions is likely not commonly displayed under natural conditions, yet provides an exciting model to test whether and how epigenetic inheritance systems are modulated by natural genetic variation.

In order to pinpoint the molecular inheritance system underlying this mortal phenotype, we focused on determining the genetic basis of the quantitative variation among the C. elegans wild isolates, using both association mapping and laboratory crosses.

First, using Genome-Wide Association (GWA) in the 97 isolates, we detected one locus on chromosome III. The locus was conﬁrmed using Near Isogenic Lines between N2 (non-MRT) and JU775 (MRT, sterile after 4-5 generations at 25 C). ⇤Speaker †Corresponding author: [email protected] ‡Corresponding author: [email protected]

10 Second, we crossed the wild isolates MY10 (MRT) and JU1395 (non-MRT) to build a collection of 120 Recombinant Inbred Lines (RILs). The distribution of phenotypes in the RILs suggested that at least two loci were associated with the MRT phenotype. Genotyping of the RILs by bulk whole-genome sequencing revealed loci on chromosomes II and V. We conﬁrmed the two regions by introgression and are currently narrowing down the genetic intervals.

As a whole, the GWA and RIL analyses unravelled the complexity of the genetic bases of the natural MRT phenotype. In addition, we investigated the small RNA contents and gene expression levels over several generations in naturally Mrt and non-Mrt C. elegans isolates. (1) Ahmed & Hodgkin. 2000. Nature;(2) Ashe et al. 2012 Cell; (3) Buckley et al. 2012 Nature; (4) Andersen et al. 2012 Nature Genetics.

11 BUB-1 is at the core of antagonistic activities in the establishment of kinetochore attachments to the mitotic spindle.

1 1 1 Frances Edwards ⇤ , Nelly Gareil , Julien Dumont†

1 Institut Jacques Monod (IJM) – UniversitéParis VII - Paris Diderot, CNRS : UMR7592 – UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France

The accurate distribution of chromosomes during mitosis relies on their bi-orientation during prometaphase: the sister chromatids of replicated chromosomes are attached to microtubules ema- nating from the opposite poles of the bipolar mitotic spindle. These attachments are mediated and regulated by protein complexes assembled on the chromatids called kinetochores. The kinetochore proteins Cenp-F and CLASP are essential for chromosome bi-orientation during the ﬁrst embryonic division of Ceaenorhabditis elegans (Cheeseman et al, Genes and Dev 2005): their depletion leads to chromosome attachment to a single spindle pole. Surprisingly depletion of BUB-1, which is required for the localisation of these proteins to kinetochores, doesn’t impair chromosome bi- orientation. Here, we show that co-depletion of BUB-1 with Cenp-Frescues chromosome bi-orientation. This suggests that BUB-1 both inhibits bi-orientation and recruits Cenp-Fand CLASP to relieve this inhibition, leading us to explore the molecular basis for these two antagonistic activities. Our results link the inhibitory activity of BUB-1 to the kinetochore localisation of its kinase domain, but not to its kinase activity. Furthermore, we ﬁnd that BUB-1 doesn’t inhibit bi-orientation via the known kinetochore microtubule-binding complexes NDC-80, RZZ and KNL-1. Regarding the functions of Cenp-F and CLASP in relieving BUB-1’s inhibition, we proceed to a structure-function analysis of Cenp-F and reveal independent roles for Cenp-F and CLASP in regulating kinetochore-microtubule attachments. Together, our results show that BUB-1 has novel functions in regulating kinetochore-microtubule attachments and that it acts synergistically with its downstream partners Cenp-F and CLASP in a new pathway to establish bi-oriented connections of chromosomes to the mitotic spindle.

⇤Speaker †Corresponding author: [email protected]

12 Role of acto-myosin cytoskeleton in cell invasion in Caenorhabditis elegans

1 2 2 1 Rodrigo C´aceres ⇤† , Laura Kelley , David Sherwood , Julie Plastino

1 Institut Curie, Paris, France – Institut Curie – 11, rue Pierre et Marie Curie 75231 Paris cedex 05 FRANCE, France 2 Duke University, Durham, USA – Department of Biology Duke University Box 90338 Science Drive Durham, NC 27708, United States

Basement membrane (BM) is a dense 2-dimensional sheet of specialized extracellular matrix that separates epithelia from underlying tissue. The penetration of cells through BM barriers, called ”invasion”, is an important process during normal tissue development and in cancer metastasis. Much has been understood concerning the genetics and signaling of how holes are formed in the BM during invasion. However less is clear about the physical forces involved: how myosin contractility participates in BM removal and how di↵erent actin polymerization factors and crosslinkers contribute to the invasive process. In particular it remains an open question as to how physical forces compensate for the loss of matrix metalloproteases (MMPs), which participate in invasion by digesting the BM, and have been unsuccessfully targeted in cancer therapy. To address these questions, we studied anchor cell (AC) invasion in Caenorhabditis elegans in wild type worms and in worms deleted for the five main MMPs of the worm genome (MMP-). AC invasion took place in MMP- worms, but with a time delay. Since it had been reported that cancer cells up- regulate myosin contractility to invade in the absence of proteases, we first hypothesized that myosin was enabling AC invasion in MMP- worms. However an RNAi screen against di↵erent components of the myosin machinery gave no enhancement of the invasion defect in MMP- worms, suggesting that myosin was not playing an important role. In addition visualization of the actin cytoskeleton in MMP- worms revealed that actin was concentrated in the AC protrusion and barely detectable in the cortex, making it unlikely that myosin contraction of the cortex was helping the cell squeeze through the BM. The enrichment of actin in the AC protrusion of MMP- worms led us to ask what actin nucleators or crosslinkers could be important in protrusion formation. RNAi against di↵erent candidates in the MMP- worms showed an invasion block upon knock-down of actin polymerization nucleators, the Arp2/3 complex and formins, and the actin crosslinker filamin. Expression of a dominant negative form of an Arp2/3 complex activator specifically in the AC led to a complete block in invasion, confirming the importance of Arp2/3 complex-based actin polymerization for AC invasion. All together these results suggest a strong dependence on branched actin filaments formed by Arp2/3 complex nucleation during AC invasion and a negligible role for acto-myosin contractility, even in the absence of proteases.

⇤Speaker †Corresponding author: [email protected]

13 Rotating and elongating embryos: SPIM microscopy reveals how C. elegans embryos extend through a ratchet mode

1 1 2 3 Xinyi Yang ⇤ , Teresa Ferraro , Julien Pontabry , Nicola Maghelli ,Lo¨ıc 3 4 3 1 Royer , Stephan Grill , Gene Myers , Michel Labouesse†

1 Institut de Biologie Paris-Seine (IBPS) – Institut de Biologie Paris-Seine – 7 Quai Saint-Bernard, 75005 Paris, France 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) – CNRS : UMR7104, Inserm : U964, universitéde Strasbourg – Parc D’Innovation 1 Rue Laurent Fries - BP 10142 67404 ILLKIRCH CEDEX, France 3 Max Plank Institute of Molecular Cell Biology and Genetics (MPI-CBG) – Pfotenhauerstraße 108, 01307 Dresden, Germany 4 Technique University Dresden (TUD) – Mommsenstraße 9 Dresden, Germany

The process of morphogenesis in C. elegans embryos is largely driven by epidermal cells. Unlike Drosophila and zebraﬁsh, no cell division or cell rearrangement is involved in C. elegans morphogenesis. Epidermis shape changes, which are characterized by junction lengthening along the anterior/posterior (A/P) direction, play a key role in this process. The nature of and the mechanical forces stimulating junction lengthening, as well as the cellular mechanisms involved are the main objectives of this project. Our lab observed that junction elongation along A/P direction increases after muscle becomes active, and fails in muscle defective embryos. To better understand which role muscles play in polarized junction lengthening, we examined the global and local movement patterns of embryo using Single Plane Illumination Microscopy, focusing on epidermal adherens junctions and muscle nuclei. We found that wild-type embryos rotated strongly soon after muscle became active, and equally frequently to an outward or inward direction. However, muscle defective and Rho-kinase mutant embryos, which stop elongation at the 2-fold stage, scarcely rotated, suggesting that rotations are important for embryo elongation. By comparing the changes of cell aspects between each seam cell, we observed that the head, body and tail mechanically behaved as partially independent entities. We next sought to understand how such movements could account for the polarized junctions lengthening, keeping in mind that C. elegans embryos are radially symmetric. By measuring the distance between two dorsal or ventral muscle nuclei, respectively, we found that dorsal and ventral muscles mostly contract alternatively, accounting for embryo rotations. Analysis of junction roughness showed that junctions along the A/P direction were stretched when seam cells were positioned outwards during embryo rotations. Our laser ablation experiments proved that these junctions were under higher tension when stretched during embryo rotations. These results depicted that asymmetric muscle activity deﬁnes the source of polarity in C. elegans embryo and provides the local driving force of epidermis stretching. We are currently testing how it impacts on the insertion of new E-cad molecules during junction remodeling.

⇤Speaker †Corresponding author: [email protected]

14 Coordinated morphogenesis through tension induced planar polarity

Ghislain Gillard 1, Oph´elie Nicolle 1, Mathieu Pinot 1, Sylvain Prigent 2, 1 Gr´egoire Michaux ⇤

1 Institut de Génétique et Développement de Rennes (IGDR) – CNRS : UMR6290, Universite de Rennes 1 – Facultéde Médecine - CS 34317 2 Av du Professeur LéonBernard 35043 Rennes Cedex, France 2 Biogenouest (Biogenouest) – Biogenouest – Rennes, France

Understanding how tissues from di↵erent developmental origins interact to achieve controlled and coordinated morphogenesis at the level of a whole organism has been mostly studied through the prism of signalling pathways controlling the activity of transcription factors. However it is likely that other mechanisms exist and we have used the morphogenetic step of C. elegans embryonic elongation to investigate this question. The ﬁrst step of elongation is achieved by cell shape changes in the lateral epidermis under the control of actomyosin dynamics. A second step then requires muscle contractions whose mechanical signal is translated into a biochemical signal in the dorsoventral epidermis to contribute to the maturation of tendon-like structures called C. elegans hemidesmosomes (CeHD; Zhang et al, Nature, 471-99). However we do not know if and how muscle contractions have an impact on the lateral epidermis. We found that muscles contractions and dorsoventral CeHD are required to establish an axial planar polarity in lateral cells by restricting the apical PAR module to junctions between lateral cells while it is absent from the junctions with dorsoventral cells. Planar polarised PAR-3 then controls actin planar organisation in lateral cells, thus determining the elongation axis of the whole embryo. Finally preliminary experiments suggest that the planar polarity of lateral cells is required for the stability of dorsoventral CeHD. We concluded that muscle contractions, relayed by dorsoventral CeHD, are required to control the planar polarity of the lateral epidermis which then contributes to CeHD maturation. This trans- tissular biomechanical feedback loop therefore contributes to achieve a coordinated morphogenesis of the C. elegans embryo.

⇤Speaker

15 A spectrin/PAK/actin-capping network stabilizes cell shapes in a morphogenetic ratchet process during C. elegans elongation

1 2 1 2 Gabriella P´asti , Alicia Lardennois ⇤ , Julien Pontabry , Teresa Ferraro , 1 2 1 1,2 David Rodriguez , Flora Llense , Christelle Gally , Michel Labouesse†

1 Institut de génétique et de biologie moléculaire et cellulaire (IGBMC) – Institut de Génétique et de Biologie Moléculaire et Cellulaire – Development and Stem Cells Department, IGBMC – CNRS UMR 7104/ INSERM U964/ Universitéde Strasbourg, 1 rue Laurent Fries, 67400 Illkirch, France 2 Institut de Biologie Paris Seine (IBPS) – Institut de Biologie Paris-Seine – UniversitéPierre et Marie Curie, IBPS, CNRS UMR7622, 7 Quai St-Bernard, 75005 Paris, France

Embryonic morphogenesis refers to changes in the shape and position of cells within the embryo. While it is clearly established that mechanical forces induced by molecular motors play a critical role in driving these changes, proper understanding of how cells respond to such forces is lacking. In particular, the interplay between cell elasticity and the response to forces remains unclear. To address those issues, we are studying elongation of C. elegans embryos, which is initially driven by actomyosin contractility till muscle becomes active at the 1.7 fold stage, and then requires muscle activity to reach the 4-fold stage. Our main goal is to understand how the epidermis, an elastic material, maintains its shape through cycles of muscle contractions, while extending only in the anterior-posterior direction. We previously established that tension resulting from repeated muscle contractions directly influences junction and cytoskeleton remodeling through a mechanotransduction pathway. Previous work has positioned the kinase PAK-1 at the crossroads of hemidesmosomes and cytoskeleton remodeling. To better understand how PAK-1 acts, we performed a yeast two-hybrid screen and an RNAi screen in a strong pak-1 mutant background, which identified one gene in common encoding the ↵-spectrin SPC-1. Using genetic tools and live imaging approaches, we further investigated the role of ↵-spectrin and its interaction with the kinase PAK-1. We found that pak-1(-) spc-1(-) mutant embryos elongate up to 1.5-fold stage and then retract to their initial shape. At the subcellular level, circumferential actin filament bundles appear thinner, overall discontinuous and not as well oriented perpendicular to the seam-dorsal junction, in a manner that is statistically di↵erent than what is observed in single pak-1(- ) or spc-1(-) defective embryos. To understand at the molecular level how these defects occur their relationship to a possible locking mechanism, we performed another RNAi enhancer screen in a spc- 1(Ø) background. We identified three actin-binding proteins, whose absence, combined with the lack of the spectrin cytoskeleton, also induces embryos to retract. Interestingly, one of them is a formin with actin bundling properties. Specific expression in the epidermis of a C-ter truncated and predicted constitutively active version of this formin rescues the retraction phenotype of pak-1(-) spc-1(-) mutant embryos. We propose that the locking mechanism involves the activation of the formin downstream of mechanical inputs from the muscles, which in turn would favor actin bundling and or anchoring while the embryo elongates and thins.

⇤Speaker †Corresponding author: [email protected]

16 CRLD-1A is a membrane-associated protein disulﬁde isomerase required for acetylcholine receptor biogenesis

1 Manuela D’Alessandro ⇤ , Magali Richard , Christian Stigloher , Thomas Boulin 2, Janet Richmond , Jean-Louis Bessereau 2

1 Univ Lyon, UniversitéClaude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, Institut NeuroMyoGene, (INMG) – CNRS : UMR5310, Inserm – 16, rue RaphaëlDubois F-69622 Villeurbanne Cedex - France., France 2 Centre de génétique et de physiologie moléculaire et cellulaire (CGphiMC) – CNRS : UMR5534, Université Claude Bernard - Lyon I (UCBL) – UNIVERSITE CLAUDE BERNARD LYON 1 Bâtiment Gregor Mendel 16 rue RaphaëlDubois 69622 VILLEURBANNE CEDEX, France

Reduction of acetylcholine receptor (AChR) number at the neuromuscular junction (NMJ) is responsible for myasthenic syndromes. The mechanisms controlling AChR metabolism remain to be elucidated but are critical for the formation of NMJs and are potential targets for counterbalancing the decreased expression of AChRs at the NMJ typical of myasthenic syndromes. C. elegans body-wall muscles are innervated by cholinergic motoneurons. Levamisole, a worm-speciﬁc cholinergic agonist, causes muscle hypercontraction and kills the worms. CRLD-1 was identiﬁed in a screen for mutants partially resistant to levamisole and was uncharacterized in C. elegans. Crld-1 is the ortholog of the Creld1 and Creld2 human genes. Creld1 mutations are associated with developmental cardiac defects and Creld2 is implicated in endoplasmic reticulum stress through a putative protein disulphide isomerase (PDI) activity. We observed a decreased number of levamisole-sensitive AChRs (L-AChRs) in crld-1 mutants and found that CRLD-1 is an ER protein that selectively regulates L-AChR expression. We demonstrated that CRLD-1 interacts with the UNC-29 subunit of L-AChRs and regulates the stability of unassembled L-AChR subunits potentially via its evolutionarily conserved PDI function.

⇤Speaker

17 Mitochondria dynamics modulation and WAH-1 (AIF homolog) diminution reduce physiopathological muscle degeneration in C. elegans.

1 2 2 Charlotte Scholtes ⇤† , St´ephanie Bellemin , Edwige Martin , Ma¨ıt´e Carre-Pierrat 3, Marie-Christine Mariol 2, Bertrand Mollereau 1, Kathrin 2 1 Gieseler‡ , Ludivine Walter§

1 Laboratoire de Biologie et modélisation de la cellule (LBMC) – CNRS : UMR5239, Inserm : U1210, UniversitéClaude Bernard-Lyon I - UCBL (FRANCE), Ecole´ Normale Supérieure - Lyon – Laboratoire de Biologie et Modélisation de la Cellule UMR 5239 - U1210 - UCBL - ENS 46 alléed’Italie 69364 Lyon Cedex 07 FRANCE, France 2 INMG (INMG -Biologie et pathogenèsedu muscle) – CNRS : UMR5310 – Institut NeuroMyoGène CNRS UMR 5310 - INSERM U1217 - UniversitéClaude Bernard Lyon 1 16 rue RaphaëlDubois 69100 Villeurbanne France, France 3 Plateforme ”Biologie de Caenorhabditis elegans” – CNRS : UMS3421 – UniversitéClaude Bernard - Lyon I bâtiment G. Mendel 16, rue RaphaëlDubois 69622 Villeurbanne Cedex, France

Mitochondria are double-membrane subcellular organelles that exercise metabolic functions including energy production. Their shape changes continually through the combined actions of fission and fusion rendering mitochondria network very dynamic. Mitochondrial fission and fusion processes are both specifically and finely regulated by GTPases of the dynamin family that are well conserved between species. In particular, fission is mediated by DRP-1, inner membrane fusion by EAT-3, outer membrane fusion by FZO-1. Mitochondria are largely implicated in pathologies and several studies show that mitochondria dynamics is disrupted during muscle degeneration. Currently, the molecular mechanisms of muscle degeneration remain poorly understood. To study this, we choose dystrophin dependent muscle degeneration similarly to the one that takes place in Duchenne Muscular Dystrophy (DMD). DMD, the most frequent myopathy, is caused by mutations in the dystrophin gene that lead to progressive muscle degeneration reflected by muscle weakness and dramatic loss of muscle cells. The Caenorhabditis elegans mutant dys-1(cx18);hlh-1(cc561ts) is used as a DMD model with progressive dystrophin-dependent muscle degeneration.

Combining the facility of mitochondria observations in alive worms and the facility to quantify muscle degenerations in C. elegans, my goal is to shed light on the role of mitochondria dynamics in the cellular mechanisms leading to muscle degeneration. dys-1(cx18);hlh-1(cc561ts) mutants exhibit perturbations of mitochondria dynamics in muscle cells. When acting on mitochondria dynamics with a null mutation drp-1(tm1108), gene of ﬁssion, or with

⇤Speaker †Corresponding author: [email protected] ‡Corresponding author: [email protected] §Corresponding author: [email protected]

18 an overexpression of eat-3 and fzo-1, genes of fusion, mitochondrial fragmentation can be reduced compared to dys-1(cx18);hlh-1(cc561ts) mutants. Furthermore, these 3 conditions exhibited reduced muscle degeneration and increased mobility. DRP-1 is also well known to be implicated in cell death processes. In C. elegans, the pro-apoptotic function of DRP-1 is dependent on its cleavage by the CED-3 caspase. We generated transgenic lines expressing DRP-1 with mutated CED-3 cleavage site. Interestingly, our data suggest that DRP-1 cleavage by CED-3 is required for DRP-1 to decrease muscle degeneration. Altogether, the pro-fission and pro-apoptotic protein DRP-1 seems to be implicated in pathological muscle degeneration. We found also that knock-down by RNAi of wah-1 (AIF homolog) can reduce both muscle degeneration and mitochondrial fragmentation of DMD mutants. Our findings provide the first indication that WAH-1 acts on mitochondrial morphology in muscle cells. Moreover, reducing WAH-1 in the absence of DRP-1 leads to a stronger decrease of dystrophin-dependent muscle degeneration than wah-1 RNAi or drp-1(tm1108) mutation alone suggesting at least in part distinct molecular pathways.

19 EPIDERMAL RESPONSE TO FUNGAL INFECTION AND WOUNDING IN C. ELEGANS

1 1 1 1 Clara Ta↵oni ⇤ , Jonathan Ewbank , Didier Marguet , Nathalie Pujol†

Sterile wounding of Caenorhabditis elegans or its infection by the fungus D. coniospora leads to a rapid increase in the transcription of antimicrobial peptide (AMP) genes in the epidermis. Performing several forward and reverse genetic screens, we have previously identiﬁed many of the players involved in this innate immune response [1]. This includes the G Protein Couple receptor DCAR-1 and its ligand HPLA that act upstream of the well conserved p38 MAPK pathway. SNF-12, a member of the SLC6 family of bioamine transporters, is another key molecule required for the induction of AMPs and was previously described to localize into vesicles. SNF-12 interacts physically with the STAT-like TF STA-2, whose activation leads to the expression of AMPs [2]. To characterise further the response of the epidermal cell, we are monitoring in vivo the subcellular localization and dynamics of these signalling proteins upon wounding. We are using spinning disc microscopy and endogenous knock-in, single insertion or classical transgenic strains to observe the response to a laser wound. This produces an immediate calcium wave (4 sec) and the formation of an actin ring (30 min) [3]. The DCAR-1 receptor, which localizes apically in the epidermal syncytium, accumulates at the wound site on a similar time-scale, in 30 min. Remarkably, SNF-12::GFP vesicles are recruited rapidly (4 min) in a directional fashion toward the wound. Microtubule (MT) bundles, observed with TBB-2::GFP, locally reorganize. Moreover, EBP-2, a protein that binds to the + end of growing MTs is rapidly (3.5 min) recruited to the wound site, preceding the formation of the actin ring. This suggests that MT reorganization coordinates the clustering of signaling proteins like SNF-12 and the actin ring formation required for wound repair.

We are now using drugs and genetic tools (spastin) to depolymerize MTs and look at the impact on the recruitments of SNF-12 and MT + end proteins and assess their role in the induction of the immune response.

1. Ta↵oni, C. and N. Pujol, Mechanisms of innate immunity in C. elegans epidermis. Tissue Bar- riers, 2015. 3(4): p. e1078432.

2. Dierking, K., et al., Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity. Cell Host Microbe, 2011. 9(5): p. 425-35. 3. Xu, S. and A.D. Chisholm, A Galpha(q)-Ca(2+) signaling pathway promotes actin-mediated epidermal wound closure in C. elegans. Curr Biol, 2011. 21: p. 1960-1967.

⇤Speaker †Corresponding author: [email protected]

20 Exploring fungal virulence using C. elegans

1 1 1 1 Xing Zhang ⇤ , Le He , Nathalie Pujol , Jonathan Ewbank†

1 Centre d’Immunologie de Marseille - Luminy (CIML) – Universitéde la Méditerranée- Aix-Marseille II, CNRS : UMR7280, Inserm : U1104 – Parc scientifique et technologique de Luminy - 163, avenue de Luminy - Case 906 - 13288 Marseille cedex 09, France

We are using the interaction between Caenorhabditis elegans and the nematophagous fungi Drech- meria coniospora as a model system to investigate fungal pathogenesis and the host response to infection. As a first step in the study of fungal virulence, we have identified candidate proteins though a combination of bioinformatic and biochemical approaches. Using transcriptome data for di↵erent stages of D. coniospora’s life-cycle, we identified genes that are preferentially expressed during infection. We are focusing on those that encode proteins that are potentially secreted into the host and could interfere with host cell signalling. Using a polyethylene glycol-mediated transformation method, we are able to make transcriptional and translation reporter strains for chosen candidates. This will allow us characterize fungal virulence factors in an in vivo setting. In parallel, we will use co-immunoprecipitation and Y2H screens to identify host proteins that interact specifically with selected fungal e↵ectors.

⇤Speaker †Corresponding author: [email protected]

21 Channel Nucleoporins recruit the Polo-like kinase PLK-1 to Nuclear Pore Complexes in prophase to direct Nuclear Envelope Breakdown

1 1 2 Lisa Martino ⇤ , Stephanie Morchoisne-Bolhy , Dhanya Cheerambathur , Lucie Van Hove 1, Julien Dumont 1, Nicolas Joly 1, Arshad Desai 2, Val´erie 1 1 Doye , Lionel Pintard†

1 Institut Jacques Monod (IJM) – UniversitéParis VII - Paris Diderot, CNRS : UMR7592 – UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France 2 Department of Cellular and Molecular Medicine (CMM) – University of California, San Diego Dept. of Cellular and Molecular Medicine 9500 Gilman Dr. MC 0651 La Jolla, CA 92093-0651, United States

In animal cells, nuclear envelope breakdown (NEBD) is required for the assembly of the mitotic spindle and for proper chromosomes segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity in space and time to trigger NEBD is still unclear. Here we show that both in human cells and C. elegans embryos, the mitotic Polo-Like kinase 1 (PLK-1) is recruited to the nuclear pore complexes in prophase, just prior to NEBD, through its Polo-Binding Domain (PBD). We identiﬁed the C. elegans nucleoporins NPP-1/Nup58, NPP-4/Nup54 and NPP- 11/Nup62, which form a trimeric complex localized at the central channel of the nuclear pore, as the critical factors anchoring PLK-1 to the Nuclear Envelope (NE). In particular, NPP-1 and NPP-11 phosphorylated on multiple Cdk1-dependent PBD-docking sites directly interact with the PLK-1 PBD. Finally, we provide evidences that PLK-1 localization to the NE is required for ecient NEBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the Nuclear Envelope in prophase, thereby facilitating phosphorylation of critical downstream targets.

⇤Speaker †Corresponding author: [email protected]

22 Mechanisms of immune activation after cuticle damage

1 2 1 Jean-Christophe Lone ⇤ , Jonathan Ewbank , Nathalie Pujol†

1 Centre d’Immunologie de Marseille - Luminy (CIML) – Universitéde la Méditerranée- Aix-Marseille II, CNRS : UMR7280, Inserm – Parc scientifique et technologique de Luminy - 163, avenue de Luminy - Case 906 - 13288 Marseille cedex 09, France 2 Centre d’Immunologie de Marseille - Luminy (CIML) – Universitéde la Méditerranée- Aix-Marseille II, CNRS : UMR7280, Inserm : U1104 – Parc scientifique et technologique de Luminy - 163, avenue de Luminy - Case 906 - 13288 Marseille cedex 09, France

We are studying the innate immune response of C. elegans to infection by a natural fungal pathogen. Through genetic and RNAi screens, we have defined several signaling pathways that lead to the activation of defense genes, like gene coding antimicrobial peptides (AMP) in the infected tissue, the adult epidermis. We were able to show that infection provokes the conversion of tyrosine to hydroxyl- phenyl-lactic acid (HPLA) and this in turn activates the G-protein coupled receptor DCAR-1 that acts upstream of a PKC-p38 MAPK pathway and the STAT-like transcription factor STA-2. Not only are dcar-1 mutants deficient in their response to infection, but they also fail to switch on AMP expression following physical injury. Thus HPLA appears to be a damage-associated molecular pattern (DAMP), and DCAR-1 to be the first DAMP receptor identified in C. elegans [1-3]. Just how infection or injury leads to an increase in HPLA is, however, unknown. We are currently dissecting the mechanism leading to its production by doing a genetic screen. Interestingly, we found that mutants lacking a specific structure on the cuticle that delineates the annuli, the furrows, exhibit a high constitutive expression of AMP genes. We know for one of them, dpy-10, that the level of HPLA is elevated, and for all of them that the high AMP gene expression is dependent on dcar-1. Thus, we designed a genetic screen in a furrow mutant (dpy-7) in which a AMP reporter inducible by infection is constitutively turned on, from the early larval stage. To avoid isolating mutants for components downstream of the GPCR, the strain used also expresses a constitutively active G↵ protein (GPA-12) specifically in the adult epidermis. Screening for mutants that block reporter gene expression in larvae but not in adults will identify genes acting upstream of GPA-12 and hence potentially reveal the initial steps leading to the production of HPLA.

We have recently screened over 60,000 genomes and obtained 5 candidate mutants. We are now further phenotypically characterising them before using whole-genome sequencing to identify the underlying molecular lesion.

1. Dierking, K., et al., Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity. Cell Host Microbe, 2011. 9(5): p. 425-35.

2. Pujol, N., et al., Distinct innate immune responses to infection and wounding in the C. elegans epidermis. Curr Biol, 2008. 18(7): p. 481-9.

⇤Speaker †Corresponding author: [email protected]

23 3. Zugasti, O., et al., Activation of a G protein-coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans. Nature immunology, 2014. 15(9): p. 833-8.

24 Fonctional study of Katanin, a prototype of microtubule-severing enzyme essential for C.elegans meiosis

1 1 2 2 Nicolas Joly ⇤† , Lisa Martino , Emmanuelle Gigant , Julien Dumont , Lionel Pintard 3

1 Institut Jacques Monod (IJM) – UniversitéParis VII - Paris Diderot, CNRS : UMR7592 – Cell Cycle and Development Team UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France 2 Institut Jacques Monod (IJM) – CNRS : UMR7592 – Cell Division and Reproduction Team UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France 3 Institut Jacques Monod (IJM) – CNRS : UMR7592 – Cell Cycle and Development Team UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France

Microtubules (MTs) are dynamic cytoskeletal polymers with instrumental functions in cell division (meiosis and mitosis), morphogenesis, motility and signaling. MTs constantly polymerize and shrink and this dynamic behavior, which is critical for their function, is regulated by a large family of MT- interacting proteins. Whereas most of these proteins interact with the microtubule plus or minus ends, another class interacts with the MT lattice and severs MTs along their length, thereby controlling MTs size and density. Three evolutionarily conserved MT-severing enzymes have been identiﬁed: Fidgetin, Spastin and Katanin. Mutation of these enzymes has been linked to various defects and pathologies including developmental defects, neurodegenerative disorders such as hereditary spastic paraplegia (HSP) and the Fidget disease.

In this project, we are using use Caenorhabditis elegans Katanin as prototype of microtubule severing- enzymes. Katanin is a heterodimer composed of a catalytic subunit (p60 –MEI-1 in C. elegans) and a regulatory subunit (p80 –MEI-2), but the relative contribution of both subunits to the MT-severing activity is unclear. Moreover, the mechanism by which Katanin is interacting with and is severing MTs is currently unknown. Using complementary biochemical and genetic approaches, we provide new insights into the molecular mechanism by which Katanin severs the microtubules.

⇤Speaker †Corresponding author: [email protected]

25 An Atypical Mechanism of Chromosome Segregation in the C. elegans Oocyte

1 2 3 4 Kimberley Laband ⇤ , Marine Stefanutti , Julie Canman , Julien Dumont†

1 Institut Jacques Monod (IJM) – UniversitéParis VII - Paris Diderot, CNRS : UMR7592 – UniversitéParis Diderot, Bât. Bu↵on, 15 rue Hélène Brion, 75205 Paris cédex 13, France 2 Institut Curie – Institut Curie – Institut Curie, France 3 Columbia University (USA) – New York, United States 4 Institut Jacques Monod (IJM) – UniversitéParis VII - Paris Diderot, CNRS : UMR7592 – Batiment Bu↵on 15 rue Hélène Brion 75205 Paris cédex 13, France

Female gametes called oocytes are produced through a speciﬁc type of cell division termed meiosis. In order to produce haploid gametes, and unlike mitotic divisions of somatic cells, meiosis involves two rounds of chromosome segregation following a single round of genome replication. Accuracy of chromosome segregation during meiosis is crucial to avoiding embryonic aneuploidy that would lead to developmental defects or spontaneous abortion. Chromosome segregation relies on a microtubule-based spindle. Concomitant to spindle assembly, multi protein complexes termed kinetochores assemble on the side of chromosomes and translate microtubule dynamics into chromosomal movements. Strikingly, in the C. elegans oocyte chromosome segregation occurs in an atypical kinetochore-independent manner. The alternative mechanism used in these oocytes for chromosome segregation is, however, unknown. Here, we used a combination of high spatial and temporal resolution live imaging, laser-mediated photoablation of microtubules and targeted inhibition of key proteins to dissect this atypical mechanism of chromosome segregation in the C. elegans oocyte.

⇤Speaker †Corresponding author: [email protected]

26 Deciphering the role of Notch signaling in Y to PDA transdi↵erentiation in vivo in c.elegans.

1 Laura Vibert ⇤ , Nadine Fisher , Sophie Jarriault†

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) – CNRS : UMR7104, Inserm : U964, universitéde Strasbourg – Parc D’Innovation 1 Rue Laurent Fries - BP 10142 67404 ILLKIRCH CEDEX, France

Cell di↵erentiation is a key process in Developmental Biology as it is essential during organism development but it is also involved in cancer when stable cell di↵erentiation is dysregulated. Manipulation of the cell di↵erentiation process has seen great progress the last ten years leading to the demonstration, in vitro, of the capacity of cells to dedi↵erentiate by forcing the expression of specific transcription factors in di↵erentiated cells. However whereas the transcription factors used for pluripotent reprogramming has led to the successful reprogramming of all cell types reported, intriguingly, several studies showed that direct reprogramming induced by one or more transcription factors is only fully e↵ective in a restricted number of cell types, i.e is cell context-dependent. Thus, as cells could di↵er in their abilities to be reprogrammed by a given inducing method, some cells appear more permissive to reprogramming. Here we have observed an exceptional in vivo event of single cell identity change during c.elegans development which involves cell di↵erentiation, cell dedi↵erentiation and cell reprogramming. We aim to go further in the understanding of cell di↵erentiation plasticity by deciphering the genetic regulatory mechanisms underlying this process. More precisely, the single Y to PDA transdi↵erentiation allows the rectal Y cell to go through a process of acquisition of a new state of potentiality and to di↵erentiate into another ectodermic cell type, a motoneuron (PDA). The dramatic changes observed in expression profiles suggest that these changes are driven by activation of key regulators which would allow the rapid switch from a cell state to the next one. Previous work in the laboratory has suggested that the Y cell acquires the competence to change its identity after its birth in the embryo, rather than inheriting it during its lineage history. Importantly Y is competent for cell transdi↵erentiation, but its neighbouring rectal cells are not. One key di↵erence between the Y cell and the other rectal cells that do not change their identity is that the sole Y cell expresses the lin-12/Notch receptor. This expression appears to be dynamic and restricted to the embryogenesis stage. Importantly, we found that a pulse of the LIN-12/Notch receptor activity is required to endow Y with the competence to change identity, and sucient to convert another cell, that we have identified as DA9, into a PDA neuron. The major aim of the project is to identify the target genes that Notch uniquely activates in the Y cell and that may be involved in setting up the competence to reprogram.

⇤Speaker †Corresponding author: [email protected]

27 Actin cytoskeleton in cell invasion and migration in Caenorhabditis elegans vulval development

1 1 1 Nagagireesh Bojanala ⇤ , Rodrigo Caceres , Julie Plastino†

1 Physico-Chimie-Curie (PCC) – UMR 168, Institute Curie – Bˆatiment Curie 26 rue d’Ulm 75248 PARIS CEDEX 05, France

Proper organization of the actin cytoskeleton is crucial for cell migration and invasion during developmental processes and pathological conditions such as cancer cell invasion, however the mechanism by which actin dynamics is adapted to perform certain functions in di↵erent cell types in a given tissue is largely unknown. To address this question, we study actin cytoskeleton during anchor cell (AC) invasion and vulva precursor cell (VPC) migration in Caenorhabditis elegans vulval development. The AC invades the basement membrane (BM) via an actin-rich protrusion, allowing for a connection to be made between the uterus and the vulval cells. Other results in the lab indicate a role for the actin polymerization nucleator, the Arp2/3 complex, in the formation of the AC protrusion, similar to what is observed for cancer cell invasion and other cell protrusion events. In mammalian cells, it has furthermore been shown that crosslinking of actin filaments is important for the stability and mechanical properties of the protrusion. However, it is unknown if actin crosslinkers play a similar role during AC invasion. To address this, we analyzed the role of the three main actin crosslinkers in the C. elegans genome: filamin (FLN-1), ↵-actinin (ATN-1), and plastin (PLST-1) during AC invasion. RNAi against fln-1 in a sensitized background where actin dynamics was slightly compromised resulted in an AC invasion defect, suggesting that crosslinkers could play a role in AC invasion. However no invasion defects were detected for worms carrying loss-of-function mutations in individual crosslinker genes fln- 1(tm545), fln-1(ok2611), atn-1(ok84) and plst-1(tm4255). Furthermore fln-1 RNAi in atn-1(ok84) gave no invasion defect. These results suggest that crosslinkers could be acting redundantly. To counter this, we are presently creating an atn-1(ok84; plst-1(tm4255) double mutant, which we will subject to fln-1 RNAi to create conditions deficient in all three of the main actin crosslinkers in C. elegans. Evaluation of invasion under these conditions will indicate to what extent crosslinkers are necessary for AC invasion.

Using tools and approaches developed for studying AC invasion, a future project will be to study the movement of VPCs, the cells that lie underneath the BM, just below the AC. Before AC invasion, VPCs are variably positioned, but in response to AC secretion of epidermal growth factor (EGF), VPCs undergo shape changes and migrations to correctly position themselves with reference to the AC. Although signaling to the actin cytoskeleton is suspected, the mechanism of VPC shape change and motility and how it depends on the actin cytoskeleton is unknown. To address this, we will analyze actin dynamics in VPCs using the F-actin reporter, Lifeact, as we have done in AC invasion, but with the use of promoters for VPC expression. We will further apply RNAi and the dominant negative approaches that we have developed for AC invasion to test the role of di↵erent actin polymerization nucleators and crosslinking proteins in VPC positioning.

⇤Speaker †Corresponding author: [email protected]

28 Overall by studying and comparing actin dynamics in AC invasion and VPC migration, we will acquire a better understanding of how actin dynamics is regulated in di↵erent cell types to perform di↵erent morphological programs for correct organ development.

29 Cellular innovations at the origin of pseudogamy in nematodes

1 2 3 4 Manon Grosmaire ⇤ , Caroline Launay , Mark Blaxter , Marie-Anne F´elix , 2 Marie Delattre†

1 Laboratoire de Biologie et Modélisation de la Cellule (LBMC) – CNRS : UMR5239, Ecole´ Normale Supérieure (ENS) - Lyon – ENS de Lyon 46 alléed’Italie 69364 LYON Cedex 07, France 2 Laboratoire de Biologie et Modélisation de la Cellule (LBMC) – CNRS : UMR5239 – ENS de Lyon 46 allée d’Italie 69364 LYON Cedex 07, France 3 Univesity of Edinburgh – University of Edinburgh Edinburgh EH9 3JF United Kingdom, United Kingdom 4 Institut de Biologie de l’Ecole´ Normale Supérieure (IBENS) – CNRS : UMR8197 – Département de Biologie Ecole´ Normale Supérieure 46, Rue d’Ulm 75005 Paris, France

Pseudogamy is a mode of reproduction in which the fertilization of an oocyte by a sperm cell is necessary even tough the male DNA does not contribute to the zygote’s genome. In the nematode species Mesorhabditis belari, embryos in which the sperm DNA does not decondense always give rise to females, while the contribution of sperm DNA allows the production of rare males in the population. It has been proposed that oocytes experiencing incomplete meiosis will not utilize the male DNA, while oocytes undergoing the regular two steps of meiosis allow the decondensation of the male DNA (Nigon, 1942). These observations suggest that pseudogamy is the consequence of a stochasticity in the progression through meiosis, accompanied by a control of the paternal DNA. Our goal is to explore the cellular innovations that have conditioned the emergence of this repro- ductive strategy within the Mesorhabditis genus. To this end, we have re-isolated 42 new strains of Mesorhabditis: 14 have a classic male/female mode of reproduction (named gonochoristic) and 28 are pseudogamous. A ﬁrst phylogeny has been obtained by sequencing of ITS, 18S and 28S revealing that pseudogamous species constitute a monophyletic group. Combined with genetic crosses and morphological description, we have so far identiﬁed 2 gonochoristic species and 10 independent pseudogamous species, and several strains per species.

The JU2817 pseudogamous strain of the Mesorhabditis belari species, isolated in Orsay, has been selected as our reference strain. Its genome, of about 200 Mb, has been recently sequenced and assembled de novo. Two other strains from this species will serve as polymorphic strains for further genetic studies. Their DNA is currently being sequenced in order to identify SNPs. The RNAs of JU2817 and of the closest gonochoristic strain M. longespiculosaDF5017 are currently being sequenced for a comparative transcriptomic analysis. In parallel we are performing a comparative cytological analysis between M. belari and M. longespicu- losa.Firstresultsshowadi↵erence in the number of chromosomes and in the progression through female meiosis.

⇤Speaker †Corresponding author: [email protected]

30 Characterization of the SET1/MLL complexes in C.elegans

1 1 1 2 Flore Beurton ⇤† , C´ecile Bedet , Matthieu Caron , J´erome Govin ,Yohann 2 1 1 1 Coute , David Cluet , Martin Spichty , Francesca Palladino‡

1 Laboratoire de Biologie et Modélisation de la Cellule UMR5239 (LBMC, UMR5239, ENS Lyon) – Ecole´ Normale Supérieure - Lyon – 46 alléed’italie 69007 Lyon, France 2 Exploring the Dynamics of Proteoms Laboratoire de Biologie àGrande Echelle (BGE) UMR 1038Inserm/CEA/UGA(EDY Plaboratory) S Commissariatal` 0EnergieAtomiqueetaux´ EnergiesAlternatives´ (CEA) Grenoble CEAGrenoble17,ruedesMartyrs38054GrenobleCedex9,France

Methylation of histone H3 Lys4 (H3K4me) is associated with active transcription in all species, and is catalyzed by highly conserved multiprotein complexes known as Compass in yeast or SET1/MLL in mammals. Biochemical analysis has shown that the H3K4 HMT activity of SET1/MLL proteins relies on protein-protein interactions within large multisubunit complexes. The composition of SET1/Compass complexes has been described in yeasts, plants, drosophila and mammals: in addition to the catalytic SET1 subunit, these complexes share in common ASH2, RbBP5, WDR5, DPY30, which are also components of MLL related complexes, and the CXXC Zinc ﬁnger protein CFP1, which is unique to SET1 complex. However in C. elegans, the biochemical composition of the complex has not been described, although single homologs of all the subunits are conserved and have shown to contribute to global H3K4 methylation.

In order to purify the SET1 complex from C. elegans embryos, and identify additional proteins associated with the complex, we undertook a proteomic approach: we used strains expressing either GFP tagged CFP-1 (from J.Ahringer’s lab) or HA tagged WDR-5.1. Immunoprecipitation experiments followed by tandem MS/MS identified all common subunits of the SET1/MLL complexes in both CFP-1 and WDR-5.1 immunoprecipitations. Interestingly, while CFP-1 copurified uniquely with SET-2, WDR-5.1 also copurified with components of the MLL-related complex, including SET-16, the histone H3K27 demethylase UTX-1, and PIS-1. Our results are consistent with the presence of both SET1 and MLL related complexes in C. elegans embryos. Furthermore, new candidates were also identified as top hits in WDR-5.1::HA and CFP-1::GFP immunoprecipitation experiments and mutants for those candidates have phenotypes in common with cfp-1 mutant.

⇤Speaker †Corresponding author: ﬂ[email protected] ‡Corresponding author: [email protected]

31 Post-transcriptional histone modiﬁcations maintain cell identity and genome stability in the Caenorhabditis elegans germline

1 2 1 1 Marion Herbette ⇤ , Marine Mercier , Fanny Michal , Claire Burny ,David Cluet 1, Ga¨elYvert 1, Hanh Nguyen 3, Monique Zetka 3, Florence Couteau 4, 1 1 Valerie Robert , Francesca Palladino†

1 Laboratoire de Biologie et Modélisation de la Cellule UMR5239 (LBMC, UMR5239, ENS Lyon) – Ecole´ Normale Supérieure - Lyon – 46 alléed’italie 69007 Lyon, France 2 Centre de génétique et de physiologie moléculaire et cellulaire (CGphiMC) – CNRS : UMR5534, Université Claude Bernard - Lyon I (UCBL) – UNIVERSITE CLAUDE BERNARD LYON 1 Bâtiment Gregor Mendel 16 rue RaphaëlDubois 69622 VILLEURBANNE CEDEX, France 3 Department of Biology - McGill University – 1205 Avenue Docteur Penfield, Montreal, QC, H3A 1B1, Canada 4 Centre de Recherche Hôpital Maisonneuve-Rosemont – 5415, boulevard de l’Assomption Montréal(Québec) H1T 2M4, Canada

Maintaining the integrity of genetic information across generations is essential for both cell survival and reproduction, and requires the timely repair of DNA damage. Specific post-translational modifications of histone tails play a central role in the DNA repair process through the recruitment of proteins and complexes with specific enzymatic activities, or by altering the chromatin state at the site of DNA lesions. The conserved Set1 histone methyltransferase (HMT) catalyzes methylation of histone H3 on Lysine 4 (H3K4), a histone modification universally associated with actively transcribed genes. Less is known about the role of Set1 family proteins in the DNA repair process. Here we show that SET-2, the Caehnorabditis elegans orthologue of Set1, is required to preserve genome integrity over subsequent generations. set-2 mutant animals show a transgenerational increase in sensitivity to DNA damage that is accompanied by a defect in the resolution of DNA-damage induced double-strand break (DSB) and chromosome fragmentation. In meiosis, absence of set-2 also results in a transgenerational increase in programmed DSBs. The defect in the repair of DSBs is not due to a failure to activate the DNA damage response (DDR) that allows detection, signaling and repair of DNA lesions, because cell cycle arrest and apoptosis, key components of this pathway, are eciently induced in set-2 mutant animal. Altogether, our results suggest that SET-2 plays a role in the transgenerational maintenance of genome stability and may play a direct role in the DSB repair process downstream of DDR signaling.

⇤Speaker †Corresponding author: [email protected]

32 Asymmetric cell division and the cytoskeleton in nematodes

1 2 Majdouline Abou-Ghali ⇤ , Marie Delattre , Julie Plastino

1 Physico-Chimie-Curie (PCC) – CNRS : UMR168, Institut Curie, UniversitéPierre et Marie Curie (UPMC) - Paris VI – Bâtiment Curie 11-13 rue Pierre et marie curie 75248 PARIS CEDEX 05, France 2 Physico-Chimie-Curie (PCC) – CNRS : UMR168, Institut Curie, UniversitéPierre et Marie Curie (UPMC) - Paris VI – Bâtiment Curie 26 rue d’Ulm 75248 PARIS CEDEX 05, France

The ﬁrst division of the Caenorhabditis elegans embryo is a classic example of asymmetric cell division, and much has been learned from this model concerning the role of the acto-myosin cortex in symmetry breaking and polarity establishment, and the role of astral microtubules in spindle positioning. Although they undergo similar asymmetric divisions, nematode embryos from other genera appear to be lacking many of the key characteristics observed for Caenorhabditis, including stereotypical spindle movements and cortex behavior. Additionally in parthenogenetic species, which develop without fertilization, the initial cue for symmetry breaking is unknown. In this project, we will determine how asymmetric division is achieved in three nematode species evolutionarily distant from C. elegans,including a parthenogenetic species. We will characterize the actin cytoskeleton in these embryos, evaluate cortical ﬂows and properties, measure rheological properties of the cytoplasm and perturb cytoskeleton dynamics via laser ablations and drug treatments. Results of this study will reveal conserved mechanical and molecular principles of the cytoskeleton for achieving asymmetric cell division, revealing the richness of solutions provided by evolution to solve a cell biology problem.

⇤Speaker

33 Analysis of syndecan function at the C. elegans neuromuscular junction.

1 Camille Vachon ⇤ , Jean-Louis Bessereau†

1 Institut NeuroMyoGène CNRS UMR 5310 - INSERM U1217 - UniversitéClaude Bernard Lyon 1 – Université Claude Bernard-Lyon I - UCBL (FRANCE) – Institut NeuroMyoGène CNRS UMR 5310 - INSERM U1217 - UniversitéClaude Bernard Lyon 1 16 rue RaphaëlDubois 69100 Villeurbanne France, France

The extracellular matrix (ECM) plays an essential role in the development and function of organs and tissues. Syndecan is an ECM component that belongs to the heparan sulfate proteoglycan (HSPG) family. It is composed of 3 polysaccharidic chains linked to a core transmembrane protein. In C. elegans syndecan is coded by a single gene sdn-1. It is required for normal vulva development and axonal guidance (Minniti et al., 2004) (Rhiner et al., 2005). Sugar chains are extensively modified including sulfation, acetylation and epimerisation of individual sugar residues. Recent reports suggest that some modifications are cell specific (Attreed et al., 2016) supporting the hypothesis of an HS code. HSPG are present at neuromuscular junctions (NMJ) but their synaptic functions remain uncharacterized. In C. elegans body-wall muscle cell receive excitatory and inhibitory innervation from cholinergic and GABAergic motoneurons, respectively. Ce-punctin (also known as MADD-4) is an ECM protein secreted by motoneurons in the synaptic cleft. Specific combinations of Ce-punctin isoforms trigger the clustering of acetylcholine or GABAA receptors at synaptic sites.

We used a BFP knock-in allele generated by the B´’ulow lab to detect SDN-1 and we observed that SDN-1 is present at NMJs and seems to be enriched at cholinergic neuro-muscular synapses. Using single-chain antibodies to label specific HS modifications in vivo suggests that some modifications could be prevalent at cholinergic junctions. Preliminary data indicate that sdn1 disruption might a↵ect the localization of acetylcholine receptors. Ongoing experiments and future plans will be presented at the meeting.

Attreed, M., Saied-Santiago, K., B´’ulow, H.E., 2016. Conservation of anatomically restricted gly- cosaminoglycan structures in divergent nematode species. Glycobiology 26, 862–870. doi:10.1093/glycob/cww037

Minniti, A.N., Labarca, M., Hurtado, C., Brandan, E., 2004. Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva. J. Cell Sci. 117, 5179–5190. doi:10.1242/jcs.01394 Rhiner, C., Gysi, S., Fr´’ohli, E., Hengartner, M.O., Hajnal, A., 2005. Syndecan regulates cell migration and axon guidance in C. elegans. Development 132, 4621–4633. doi:10.1242/dev.02042

⇤Speaker †Corresponding author: [email protected]

34 Identiﬁcation of novel regulators of GABAergic synaptogenesis in the nematode Caenorhabditis elegans.

1 1 1 Marine Gueydan ⇤ , Bérangère Pinan-Lucarré , Aurore-Cécile Valfort , 1 Jean-Louis Bessereau†

1 Institut Neuromyogène - CNRS UMR 5310 - INSERM U1217 (INMG) – Institut Neuromyogène – Université Claude Bernard Lyon 1 - 16 rue RaphaëlDubois 69100 Villeurbanne, France

In the central nervous system, the inhibitory system plays a key role in neuronal network excitability. Defects in the excitatory versus inhibitory balance could lead to neuropathies. To identify novel genes and mechanisms involved in the formation and the regulation of inhibitory synapses, we use the inhibitory GABAergic neuromuscular junction of the nematode Caenorhabditis elegans as a genetically tractable model. At these synapses, fast neurotransmission is ensured by type A ionotropic GABA receptors (GABAAR), which form post-synaptic clusters in front of GABA release sites. Specifically, we performed an EMS genetic screen based on the direct visualization of fluorescently tagged GABAAR in vivo in a knock-in strain. A second EMS genetic screen has been performed on this knock-in strain in a sensitized background. We identified 56 (respectively, 31 + 25) mutants with abnormal GABAAR localization among the 3648 (respectively, 1728 + 1920) mutagenized haploid genomes isolated. We used for 36 mutants a novel whole genome sequencing strategy to simultaneously map and identify mutations responsible for the GABAAR localization defect without any prior time- consuming genetic mapping. In parallel with the validation of some potential candidates, we are currently using CRISPR technology to generate a knock-out strain of an interesting candidate which could play a role in GABAAR tracking or membrane surface localization.

⇤Speaker †Corresponding author: [email protected]

35 The role of sel-10 in transdi↵erentiation

1 C´ecile Delance ⇤ , Steven Zuryn , Sophie Jarriault

1 Labo Jarriault – IGBMC – 1, rue Laurent Fries, France

Transdi↵erentiation (TD) is a process during which one di↵erentiated cell changes its identity and become another specialized cell type. In our laboratory, we study a well-characterized single cell transdi↵erentiation event which occurs naturally in the rectum of the worm. One of the 6 rectal cells, named Y, transdi↵erentiates into the PDA motor neuron (Y-to-PDA). This process begins in Y at the L1larval stage and is completed at the L3 larval stage.

In this poster, I present our study on sel-10 which is an ubiquitin ligase E3 involved in Y-to-PDA transdi↵erentiation. We found that sel-10 is genetically interacting with a histone demethylase (jmjd- 3.1 ) and also have a role positive role during TD, just as jmjd-3.1. One model is that SEL-10 ubiq- uitinates a substrate which acts as a probable repressor of Y-to-PDA TD, triggering its degradation by the proteasome. To test this hypothesis, we are examining which SCF complex is involved in Y transdi↵erentiation. In addition, we have set up di↵erent strategies, using genetics and RNAi screening approaches, to identify SEL-10 substrate(s). We will thus report on our progresses at the meeting.

A hallmark of jmjd-3.1 and sel-10 mutants is their variable Td defect penetrance and their sensitivity to stress. We hypothesised that jmjd-3.1 and sel-10 activity ultimately impacts gene expression, for example by stabilising stronger expression of gene that are key to the process. To test this hypothesis in a quantitative manner, we have been setting up smFISH experiments. Since jmjd-3.1 and sel-10 both impact the re-di↵erentiation phase into the PDA neuron, we are examining the expression of genes involved in or expressed during re-di↵erentiation, in various single and double mutants. Preliminary results will be shown at this meeting.

⇤Speaker

36 Analysis of activity-dependent synaptogenesis at the SAB neuromuscular junction.

1 2 Alexis Weinreb ⇤ , Jean-Louis Bessereau†

1 Institut NeuroMyogène (INMG) – CNRS : UMR5310, UniversitéClaude Bernard - Lyon I (UCBL), Inserm : U1217 – UNIVERSITE CLAUDE BERNARD LYON 1 Bâtiment Gregor Mendel 16 rue RaphaëlDubois 69622 VILLEURBANNE CEDEX, France 2 Institut NeuroMyoGène CNRS UMR 5310 - INSERM U1217 - UniversitéClaude Bernard Lyon 1 – Université Claude Bernard-Lyon I - UCBL (FRANCE) – Institut NeuroMyoGène CNRS UMR 5310 - INSERM U1217 - UniversitéClaude Bernard Lyon 1 16 rue RaphaëlDubois 69100 Villeurbanne France, France

In various systems, activity of neurons or muscle can shape connectivity in the network, and regulate the strength of synapses, providing the basis for learning and memory. In C. elegans,fewsystems have been reported where activity plays a role in synapse formation and function. We are investigating the e↵ects of activity on the SAB system. In this system, it has been shown that electrical silencing of the muscle cells can regulate the morphology of the SAB motoneurons (Zhao and Nonet, 2000). Using fluorescently-tagged acetylcholine receptors (AChR), we observed SAB overgrowth and ectopic synapse formation in unc-13 and unc-18 mutant worms where neuromuscular transmission was disrupted. We could confirm that this e↵ect is not due to a general loss of locomotion, as there is no SAB overgrowth in unc-54 mutants that are paralyzed. Transgene-mediated inhibition of muscle cell activity through the HisCl1 histamine-gated chloride channel and the TWK-18 temperature-dependent potassium channel also leads to SAB overgrowth, and allowed us to pinpoint a critical developmental phase. These results suggest the existence of an unknown retrograde signal. Furthermore, increasing synaptic transmission by acetylcholinesterase inhibition leads to a decrease in the number of AChRs at the NMJ, suggesting an activity-dependent regulation of the AChR number. Through a transcriptomic approach, we expect to find genes involved in the overgrowth of the SAB and the regulation of AChR number. We are using RNA-Seq to detect genes di↵erentially expressed upon electrical manipulation of the muscle cells. In parallel, we are using the tools that we developed to better define the conditions leading to SAB overgrowth and AChR downregulation, as well as testing a number of candidates genes.

References: Zhao, H., and Nonet, M.L. (2000). A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction. Development 127, 1253–1266.

⇤Speaker †Corresponding author: [email protected]

37 A Simple and rapid high throughput method to isolate large male populations.

1 1 2 1 Aniela Zablocki , Jorge Merlet ⇤† , Julie Plastino , Vincent Galy‡

1 Institut de Biologie Paris-Seine (IBPS) – Universit´ePierre et Marie Curie (UPMC) - Paris VI, CNRS : UMR7622 – 9 quai Saint-Bernard, 75005 PARIS, France 2 Institut Curie, Paris, France – Institut Curie – 11, rue Pierre et Marie Curie 75231 Paris cedex 05 FRANCE, France

C. elegans worms have two sexes: a self-fertilizing hermaphrodite (XX) and a male (X0). Males arise by spontaneous non-disjunction at very low frequency (0.1%) in the wild type hermaphrodite germ line, their frequency in populations can increase up to 50% through mating. Therefore, maintaining and purifying males in large populations can be tricky and often require the use of genetic mutants, i.e. him mutants, increasing the amount of males up to 30%. However, male isolation by single worm picking is long and fastidious and the number of males isolated is rapidly limited. In order to facilitate work involving high amounts of males we developed a protocol allowing us to easily obtain large worm populations, containing him-8(e1489) mutation, on agar plates and rapidly purify the males using a filtering method. Using Agar/peptone rich 150 mm plates we were able to grow up to 150 000 worms/plate. Growing/maintaining large population of worms on these large plates is less time consuming and much easier to handle than in liquid cultures. Then we developed a filtering method on large worm population allowing us to specifically remove eggs, larvae and hermaphrodites from the culture within 1 hour. By combining several filters sizes (40 µm and 30 µm) we were able to obtain large male populations with > 95% of purity. Our final aim is to realize genetic EMS screens involving male mating. Such screens will identify new mutants, which will help to dissect the molecular mechanisms insuring the uniparental inheritance of mitochondria.

⇤Speaker †Corresponding author: [email protected] ‡Corresponding author: [email protected]

38 Combining optogenetic and transcriptomic approaches to dissect thermal nociceptor habituation in C. elegans using LiTeSt: a versatile Light and Temperature Stimulation platform

1 Andrei-Stefan Lia ⇤

1 Universit´ede Fribourg – Av. Europe 20 1700 Fribourg, Switzerland

Nociception is a prevalent feature amongst animals that allows for the perception of pain via sensory neurons. Nociception is coupled with the downstream triggering of pain avoidance behaviours in response to a multitude of noxious environmental stimuli. As such, nociception is a crucial mechanism that has evolved to avoid or minimize damage to the organism. Our group studies thermal nociception in the nematode C. elegans, which is well suited as a model in neuroscience due to its relatively simple nervous system encompassing 302 neurons.

C. elegans presents ecient and robust noxious heat avoidance (Schild et al. 2013). However, pro- longed noxious heat stimulation (28 C) leads to a reduction in the nematode’s avoidance behaviour, mediated via CMK-1 signaling in the nucleus of the thermal nociceptor neurons called FLP (Schild et al. 2014). Nothing is known about the transcriptional targets of the CMK-1 pathway, which could mediate long term desensitization/habituation.

Therefore, we have created a C. elegans strain in which we can habituate the FLP neurons either ther- mally or optogenetically. Our aim is to use this strain to study the di↵erential expression of transcripts between unhabituated and habituated animals. This approach required us to build a novel platform called LiTeSt (Light and Temperature Stimulation), which is used to deliver optogenetic/thermal stimulation and to record worm behavior. Additionally, we have written software to automate downstream behavioural analysis. Hopefully, this transcriptomic dataset as well as the abovementioned tools will help towards a break- through in clinical pain management.

⇤Speaker

39 FRET imaging and thermalized microﬂuidics: a combined approach to monitor the C. elegans CaMK-CREB pathway in vivo

1 1 Gabriella Saro ⇤ , Dominique Glauser†

1 University of Fribourg – Chemin du Mus´ee10, Switzerland

As it provides unbeatable spatiotemporal resolution, live imaging monitoring of cellular events in response to stimuli is an extremely informative approach in experimental biology. Here, we present an imaging system combining precise microfluidic-based temperature control with epifluorescence microscopy in order to analyse specific signalling events in vivo in intact Caenorhabditis elegans expressing new genetically-encoded FRET-based probes. We apply this system to study thermal nociceptor neurons, which are specialized sensory neurons responsible for detecting and encoding noxious heat. These neurons are essential to trigger avoidance responses and prevent tissue damage. A previous genetic analysis in our lab [Schild et al. Neuron 2014] showed that the Ca2+/calmodulin-dependent protein kinase-1 (CMK-1) in C. elegans is a master switch controlling the threshold for thermal nociceptive response and is implicated in desensitization upon long term exposure to noxious heat. In order to better understand the mode of action of this kinase, it is now essential to define when and where in the cell it is activated in response to heat. Toward this goal, we established a method to monitor the spatiotemporal pattern of activity of CMK-1 in specific nociceptors in vivo. Our approach implicates two main methodological developments. First, we have designed FRET sensors based on the protein structures of CMK-1, of its regulator CKK-1 (CMK-1 kinase) and of its e↵ector CRH-1 (CREB homolog 1). Second, we have adapted a thermalized microfluidic system [CherryTemp, Rennes, France] for the implementation of precise noxious temperature stimuli on immobilized worms and dynamic ratiometric fluorescence imaging. To validate the system, we initially used Cameleon-based calcium imaging and demonstrated successful heat-evoked neural activation. Next, we used our CMK-1 kinase activity reporter and successfully engaged it to show that CMK-1 activation in the nucleus of nociceptor neurons is triggered within a few seconds of neuron activation. Ongoing work includes the characterisation of longer stimulations triggering desensitization and genetic manipulations of signalling components sup- posed to act upstream of CMK-1. So far, the combination of heat stimulation and fluorescent imaging was restricted to the use of calcium sensors like Cameleon. Our FRET-based sensor development is an innovative step toward a detailed direct investigation of other signalling pathways. We believe that this strategy can be successfully transferred to investigate a variety of biosensors and reporters to obtain physiologically or pathologically relevant spatiotemporal patterns of cell activity.

⇤Speaker †Corresponding author: [email protected]

40 Screening for nociception genes in C. elegans using an optogenetic approach

1 Filipe Marques ⇤

1 Univesity of Fribourg – Chemin du Mus´ee10, Department of Biology, University of Fribourg, Switzerland

Nociception is the process associated with the detection and transmission of noxious stimuli by nociceptors into the nervous system. Nociceptors are responsible for pain sensation and appropriate evasive, nocifensive behaviors that can prevent harm to the animal. Any advance in our understanding of the molecular substrates or properties of the neural circuits involved in nociception may therefore help developing better therapeutic approaches for pain management. Our goal is to get new insights on molecular mechanisms modulating nociception circuit function using the advantages of C. Elegans that possess nociceptors and produce a stereotyped avoidance behavior in response to noxious stimuli.

Classical genetic screens on behavioral phenotypes are limited by several factors, among which the di- culty to implement very reproducible stimuli and the existence of functionally redundant genetic/neural pathways. To circumvent these limitations, our plan is to implement an optogenetic pipeline to create a ”synthetic behavior paradigm”. The idea is to use transgenic animals expressing channelrhodopsin in speciﬁc neurons, enabling their artiﬁcial stimulation with light and the generation of light-induced behaviors; then to mutagenize these animals and screen for mutants with defective light-induced behavior. We have generated and characterized a strain expressing CoChR in the FLP thermal nociceptors that we used for the mutagenesis screen. We recovered a total of 13 new mutant lines with a reproducible, inherited phenotype at the homozygote state. We are now embarking on further characterizing the mutants and mapping of the mutations.

⇤Speaker

41 Identiﬁcation of novel regulators of the two-pore domain potassium channel EGL-23 in Caenorhabditis elegans

1 Sonia El Mouridi ⇤ , Claire Lecroisey , Alice Leclercq , Marine Mercier , Philippe Tardy , Thomas Boulin

1 Institut NeuroMyoG`ene (INMG) – Universit´eClaude Bernard-Lyon I - UCBL (FRANCE) – 8 RUE RAPHAEL DUBOIS, 69100 Villeurbanne, France

Two-pore domain potassium channels (K2P) compose a family of conserved potassium-selective ion channels responsible for the establishment and maintenance of the electrical membrane potential of metazoan cells. Despite their fundamental role, comparatively little is known about the cellular processes that control K2P channel function in vivo. In particular, we know only of few factors that directly control the number, the activity and the localization of K2Ps at the cell surface. Thus, we have performed two forward genetic screens in the nematode C. elegans to identify new regulators of the EGL-23 K2P potassium channel. To analyse the expression pattern of egl-23, we have used CRISPR/Cas9 genome engineering to generate an egl-23::SL2::TagRFP-T reporter strain. We ﬁnd that egl-23 is expressed in VM2 vulval muscles, somatointestinal, and anal depressor smooth muscles. EGL-23 is also expressed in VC4-VC5 motoneurons, in two IL2 neurons, and a dozen unidentiﬁed head and tail neurons. In addition, egl-23 is expressed in the HMC (head mesodermal cell), a cell of unknown function in C. elegans.

Gain-of-functions mutants of egl-23 are egg-laying defective (Trent et al., 1983), consistent with the expression of a hyperpolarizing EGL-23 channel in VM2 muscles. We screened 20 000 haploid genomes mutagenized with EMS and isolated 28 intragenic and 4 extragenic suppressor mutants, which were able to lay eggs again. This suppressor screen has shown that egg-laying can be restored mainly by mutation of egl-23. To circumvent this bias, we used an EGL-23-TagRFP-T CRISPR knockin strain and performed a direct visual screen for mutants with modiﬁed expression of EGL-23. In this much smaller screen (5030 mutagenized haploid genomes), we isolated 15 extragenic suppressors and only one egl-23 mutant.

To identify the causal mutation in our collection of mutants, we have combined genetic mapping and whole genome sequencing to obtain a small list of candidate genes for each strain. Using this strategy, we have identiﬁed four novel regulators of egl-23 expression: APX-1, SEL-2, SEL-12, and LIN-12.

These four genes belong to the well-known LIN-12/Notch signalling pathway. APX-1 encodes one of three C. elegans DSL proteins, ligands of Notch receptors. apx-1, lin-12 and sel-12 mutants from our screen show reduced expression of egl-23 speciﬁcally in VM2. This suggests that egl-23 might be a transcriptional target of lin-12 /Notch signalling in VM2 muscles. In contrast, SEL-2 acts as a negative regulator of lin-12 /Notch (De Souza et al., 2007) and we indeed

⇤Speaker

42 saw no change in egl-23 expression in vulval muscles. However, we identiﬁed sel-2 mutants because of a strong decrease of egl-23 expression in four of the six head neurons which are considered for our screen. We are currently performing experiment to determine if sel-2 regulates EGL-23 expression in in lin-12-dependent or independent manner.

43 wrmScarlet, seeing is believing.

1 Sonia El Mouridi ⇤ , Alice Leclercq , Thomas Boulin

1 Institut NeuroMyoG`ene (INMG) – Universit´eClaude Bernard-Lyon I - UCBL (FRANCE) – 8 RUE RAPHAEL DUBOIS, 69100 Villeurbanne, France

C. elegans is a small, fully transparent nematode making it ideal for light microscopy studies. Recent developments of CRISPR/Cas9 genome engineering have made it possible to modify virtually any part of its genome. In particular, it has opened the possibility to tag proteins of interest with fluorescent reporter genes. A multitude of fluorescence proteins with di↵erent biophysical properties are available nowadays. Thus, the challenge is to choose the best tag to be able to detect a protein of interest expressed at physiological levels in vivo. It is necessary to take into account various parameters in the choice of the fluorescent protein: the intensity, the signal-to-noise ratio and the resolution. In addition, for every new protein that is discovered and validated in vitro, in bacteria or cultured cells, it remains to be seen how it behaves in C. elegans cells and specific subcellular compartments. Here we compare the performance of three di↵erent fluorescent proteins. We have generated knock-in strains in which the potassium channel TWK-18 is fused to TagRFP-T, TagBFP or wrmScarlet.

All three knockins showed detectable ﬂuorescence at the surface of muscle cells when immobilized on glass slides and observed with a spinning disk confocal microscope equipped with a sensitive EM- CCD camera.

We also detected ﬂuorescence in twk-18::TagRFP-T when using a Nikon AZ100 macroscope coupled to a Hamamatsu Flash 4.0 CMOS camera. However, no ﬂuorescence was visible by eye.

The intensity of the fluorescent signal was similar when we compared twk-18::TagRFP-T with the TagBFP variant. Detection of blue light is however made more dicult by the strong autofluorescence of the intestine at these wave lengths. However, we noticed a marked increase in the resolution of our images, likely due to the shorter wave length of blue light. Finally, we tested for the first time wrmScarlet, a codon-optimized version of the novel red fluorescent protein mScarlet (Bindels, 2016). We quantified the fluorescence observed in our wrmScarlet strain and found that it was 5-8 times brighter than TagRFP-T. With this new fluorescent reporter, we are now able to detect TWK-18::wrmScarlet fluorescence in moving animals without fixation, using a CMOS camera or by direct observation.

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44 COULD OUR LIFE BE SWEETER AND LONGER?

1 1 1 1 Alexia Gomez ⇤† , Anne Lauren¸con , Paola Fabrizio , Anne-Laure Bulteau , Hugo Aguilaniu 1

1 Institut de Génomique Fonctionnelle de Lyon (IGFL) – CNRS : UMR5242, Institut national de la recherche agronomique (INRA) : UA1288, UniversitéClaude Bernard - Lyon I (UCBL), Ecole´ Normale Supérieure (ENS) - Lyon – Ecole Normale Supérieure de Lyon 46 alléed’Italie 69364 Lyon CEDEX07, France

It is widely known that glucose is an essential biological substrate but its excess can have extremely deleterious e↵ects on many health parameters that impact negatively lifespan as well as healthspan. Indeed, animals exposed to glucose tend to exhibit lower lifespan. This holds true for many species, including C. elegans. Serendipitously, we found a condition in which not only glucose exposure is no longer toxic but it increases lifespan strikingly. We discovered that germline less animals subjected to a drastic dietary restriction (bacterial deprivation, BD) responded positively when exposed to glucose, while in the rest of conditions tested, the addition of glucose always reduced lifespan dramatically. To better understand how the germline less glp-1(e2141ts) mutant animals under bacterial deprivation could beneﬁt from glucose exposure, we performed comparative microarray analysis on all studied nutritional conditions. Besides identifying numerous genes always induced by glucose exposure, we found that three genes all coding a putative glycerol-3-phosphate phosphatase (pgp)wereexclusively up-regulated in the condition in which glucose is no longer toxic (germline less animals subjected to BD). This is interesting because pgp, is is involved in the detoxiﬁcation of toxic glycolytic side products in mammalian cells and the activation of pgp worm homologues may contribute to prolong the lifespan of germline less animals in response to glucose.

Through a lipidomic study, we have also found that the fatty acid chains of the triacylglycerides of glp-1 mutants subjected to BD in the presence of glucose have a reduced number of carbon atoms . This phenotype may depend on modifications of the expression/activity of specific fatty acid elongases, which may be important for lifespan regulation. Together, both genome-wide expression profiling and lipidomics have provided us with important hints to start elucidating the mechanisms responsible for the unexpected pro-longevity response induced by glucose in germless food deprived worms.

⇤Speaker †Corresponding author: [email protected] List of participants

Abou-Ghali Majdouline • Beaufils Mathilde • Becker Sarah • Bedet Cécile • Berardozzi Romain • Bernon Damien • Bessereau Jean-Louis • Bidaud-Meynard Aurelien • Bojanala Nagagireesh • Boulin Thomas • Caceres Rodrigo • Caron Matthieu • Carre-Pierrat Ma¨ıté • Cecere Germano • Costache Vlad • Crist Anna • D’Alessandro Manuela • Demesmay Léa • Dumont Julien • Dupuy Denis • Edwards Frances • El Mouridi Sonia • Fabrizio Paola • Félix Marie-Anne • Fischer Nadine •

46 Flore Beurton • Fradin Chantal • Frezal Lise • Garvis Steve • Gendrel Marie • Gieseler Kathrin • Gilles Floriane • Gomez Alexia • Granger Laure • Grosmaire Manon • Grover Manish • Gueydan Marine • Herbette Marion • Hoang Dieuhuong • Jenzer C´eline • Joly Nicolas • Jospin Maelle • Katic Iskra • Laabid Driss • Laband Kimberley • Lardennois Alicia • Launay Caroline • Laurencon Anne • Laurent Patrick • Legouis Renaud • Lia Andrei-Stefan • Lone Jean-Christophe • Luccardini Camilla • Mariol Marie-Christine • Marques Filipe • Martino Lisa • Maton Gilliane •

47 Mercier Marine • Mergoud Adeline • Merlet Jorge • Michaux Grégoire • Molin Laurent • Muller Pascale • Osuna-Luque Jaime • Palladino Francesca • Parrales Valeria • Pinan-Lucarre Berangere • Plancke Camille • Plastino Julie • Prigent Séréna • Qinghao Zhang • Rapti Georgia • Rapti Georgia • Richaudeau Bénédicte • Riva Claudia • Robert Valérie • Robin Fran¸cois • Roy Charline • Saro Gabriella • Scholtes Charlotte • Solari Florence • Stratigi Katerina • Ta↵oni Clara • Tardy Philippe • Vachon Camille • Vibert Laura • Vuong Thanh • Wattin Marion • Weinreb Alexis •

48 Yang Xinyi • Zhang Xing • Zhou Xin •