Gally C et al. (2003) Med Sci (Paris) "[C. elegans: of neurons and genes]"

Gally C, Bessereau JL

[Med Sci (Paris), 2003]

The human brain contains 100 billion neurons and probably one thousand times more synapses. Such a system can be analyzed at different complexity levels, from cognitive functions to molecular structure of ion channels. However, it remains extremely difficult to establish links between these different levels. An alternative strategy relies on the use of much simpler animals that can be easily manipulated. In 1974, S. Brenner introduced the nematode Coenorhabditis elegans as a model system. This worm has a simple nervous system that only contains 302 neurons and about 7,000 synapses. Forward genetic screens are powerful tools to identify genes required for specific neuron functions and behaviors. Moreover, studies of mutant phenotypes can identify the function of a protein in the nervous system. The data that hove been obtained in C. elegans demonstrate a fascinating conservation of the molecular and cellular biology of the neuron between worms and mammals through more than 550 million years of evolution.

Gally C et al. (2003) J Neurosci "GABA is dispensable for the formation of junctional GABA receptor clusters in Caenorhabditis elegans."

Bessereau JL, Gally C

[J Neurosci, 2003]

At GABAergic synapses, GABA receptors form high-density clusters opposite GABA release sites. Whether GABA release per se plays a role in the formation of GABA receptor clusters remains uncertain. To address this question in vivo, we characterized GABA receptor clustering in the nematode Caenorhabditis elegans. In C. elegans, body wall muscles receive excitatory inputs from cholinergic motor neurons and inhibitory inputs from GABAergic neurons. Using immunohistochemistry and green fluorescent protein-tagged proteins, we observed that the muscle GABA receptor UNC-49 is precisely clustered opposite GABA release sites. During development, these clusters appear slightly after the detection of presynaptic vesicles. If motor axons are mislocalized as in unc-5 mutants, GABA receptors cluster opposite ectopic axons at GABA release sites. Together, these data imply that a motor neuron-derived factor is instructing GABA receptor clustering. Presynaptic localization of this clustering activity requires the neuronal kinesin UNC-104, suggesting that release of GABA from synaptic vesicles may represent the clustering signal. However, unc-25 mutants do not synthesize GABA but do cluster postsynaptic GABA receptors indistinguishably from the wild type. Therefore, at GABAergic neuromuscular junctions, GABA receptor clustering requires nerve-muscle interaction but not GABA neurotransmission.

Gally C et al. (2016) Methods Enzymol "Functional and Genetic Analysis of VAB-10 Spectraplakin in Caenorhabditis elegans."

Gally C, Labouesse M, Zhang H

[Methods Enzymol, 2016]

Intermediate filaments (IFs) are involved in multiple cellular processes that are essential for the maintenance of cell and tissue integrity. To achieve this crucial function, IFs have to be organized as long and resistant filaments across the cells and to be tightly anchored at the cell periphery. This anchoring takes place at the level desmosomes and hemidesmosomes through proteins belonging to the spectraplakin family. Here, we focus on the sole nematode Caenorhabditis elegans spectraplakin locus vab-10 that is essential to connect the epidermis to the cuticle apically and to the muscles basally. After briefly reviewing the structure of the gene, we first present the genetic tools available to study this gene as well as the reagents to examine the distribution of its translation products. We discuss the functional assays that enable examining their function. Finally, we detail a genetic method to identify spectraplakin functional partners through RNAi screens, and a biochemical method to examine the phosphorylation status of IFs.

Gally C et al. (2004) Nature "A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans."

Gally C, Eimer S, Richmond JE, Bessereau JL

[Nature, 2004]

Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates.

Gally C et al. (2009) Development "Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles."

Quintin S, Labouesse M, Landmann F, Wissler F, Zahreddine H, Gally C

[Development, 2009]

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4.

C Gally et al. (2004) European Worm Meeting "LEV-10 ENCODES A TRANSMEMBRANE PROTEIN REQUIRED FOR ACETYLCHOLINE RECEPTOR CLUSTERING AT NEUROMUSCULAR JUNCTIONS IN C. ELEGANS"

C Gally, J Richmond, H Ung, S Eimer, J Bessereau

[European Worm Meeting, 2004]

Clustering neurotransmitter receptors at the synapse is critical for efficient neurotransmission. In C. elegans genetic dissection of cholinergic neurotransmission has been greatly facilitated by the use of the antihelmintic drug levamisole: levamisole activates AChR receptors present at neuromuscular junctions (NMJs) which causes muscle hypercontraction and even death at high concentrations. Screens for mutants which exhibit strong resistance to levamisole have identified four genes encoding AChR subunits and two genes that are required for the biosynthesis of levamisole-sensitive AChRs. However, no genes required for AChR clustering were cloned. We hypothesised that impairing the function of such genes would generate subtle phenotype and screened for mutants only weakly resistant to levamisole. Using Mos-1 mediated mutagenesis, we identified a novel allele of lev-10. lev-10 mutants display subtle locomotory impairment and are paralysed on 1 mM levamisole. However, levamisole-sensitive AChRs are no longer detected at NMJs by immuno-fluorescence despite normal cholinergic innervation. Electrophysiological recordings of muscle cells indicate that functional AChRs are present at the muscle cell surface at similar levels in lev-10 and WT animals, but they no longer cluster at the synapse. lev-10 encodes a type I transmembrane protein carrying five CUB domains and one LDLa domain in its extracellular region. This protein is localised to cholinergic neuromuscular junctions and is required in body-wall muscles for AChR clustering. Furthermore, we demonstrate that the clustering activity of LEV-10 is contained in its extracellular region, thus suggesting a novel mechanism for AChR clustering which relies upon extracellular protein-protein interactions. Such a mechanism is likely to be evolutionary conserved since CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and may be used to cluster ionotropic receptors in vertebrates.

Christelle GALLY et al. (2001) International C. elegans Meeting "A motoneuron-derived signal is required for differentiation of post-synaptic domains at GABAergic neuromuscular junctions in C. elegans"

Jean-Louis BESSEREAU, Christelle GALLY

[International C. elegans Meeting, 2001]

Chemical synapses are formed by the apposition of two highly specialized structures i.e. the pre- and post-synaptic differentiations. In vertebrates, genes that are required for coordinate differentiation of these structures have been identified such as agrin at cholinergic neuromuscular junctions or neurexin / neuroligin and cadherin at glutamatergic synapses. However, no such genes have been identified for GABAergic synaptogenesis. We would like to identify the genes that participate in the formation of GABAergic neuromuscular junction in C. elegans using a genetic approach. Body wall muscle cells are innervated by cholinergic excitatory motoneurons and GABAergic inhibitory motoneurons. In order to analyze the relationship between pre- and post-synaptic domains, we expressed a fusion between synaptobrevin (a synaptic vesicle protein) and CFP in GABAergic motoneurons and a fusion between the GABA receptor UNC-49 and YFP in body wall muscle cells. In wild-type adults, there was a strict correlation between accumulation of pre-synaptic vesicles and clusters of GABA receptor in discrete post-synaptic domains. Furthermore, during development, no GABA receptor clusters were detected dorsally before rewiring of the DD motoneurons that innervate dorsal muscles after the L1 stage. These results suggested that a signal provided by motoneurons was triggering post-synaptic differentiation. To test this hypothesis, we expressed unc-47::snb-1-CFP ; unc-49-YFP in unc-104(e1265) . In this kinesin mutant, synaptic vesicles are no more transported in neurites and remain concentrated in cell bodies (Hall and Hedgecock, 1991). No SNB-1-CFP nor UNC-49-YFP could be detected dorsally. Ventrally, GABA receptor clusters were observed in contact with motoneuron cell bodies. Therefore, at least one neural factor seems to be transported by UNC-104 up to pre-synaptic sites and to cause formation of GABA receptor post-synaptic clusters. To characterize the role of GABA in this process, we examined UNC-49 distribution in unc-25 mutants that do not synthesize GABA. We could not see any difference between unc-25 and wild-type worms. Together with the results of Jin et al. (J Neurosc., 1999) who demonstrated that pre-synaptic differentiation is normal in unc-25 background, we concluded that GABA transmission per se is not required for GABAergic synaptogenesis. We then analyzed GABAergic synaptogenesis in unc-30 mutants. unc-30 encodes a transcription factor that controls most GABAergic features of D neurons. The number of pre-synaptic vesicle clusters was decreased much more dramatically in the ventral cord than in the dorsal cord. These results are in agreement with electron microscopy reconstruction data (John White, unpublished results). Despite the drastic reduction of synapse number in the ventral cord, we observed that UNC-49 concentrated near the cord ventrally but did not form high density aggregates. This suggests that either the few remaining GABAergic synapses or the cholinergic innervation which is not affected by unc-30 mutations are sufficient to polarize muscle cells and generate a subcellular compartment where the GABA receptors are addressed. To identify the motoneuron factors responsible for GABAergic post-synaptic differentiation, we have undertaken a genetic screen for mutants with altered UNC-49-GFP distribution.

Jarriault S et al. (2024) J Exp Biol "Developmental plasticity: a worm's eye view."

Jarriault S, Gally C

[J Exp Biol, 2024]

Numerous examples of different phenotypic outcomes in response to varying environmental conditions have been described across phyla, from plants to mammals. Here, we examine the impact of the environment on different developmental traits, focusing in particular on one key environmental variable, nutrient availability. We present advances in our understanding of developmental plasticity in response to food variation using the nematode Caenorhabditis elegans, which provides a near-isogenic context while permitting lab-controlled environments and analysis of wild isolates. We discuss how this model has allowed investigators not only to describe developmental plasticity events at the organismal level but also to zoom in on the tissues involved in translating changes in the environment into a plastic response, as well as the underlying molecular pathways, and sometimes associated changes in behaviour. Lastly, we also discuss how early life starvation experiences can be logged to later impact adult physiological traits, and how such memory could be wired.

Quintin S et al. (2016) Genesis "Non-centrosomal microtubules in C. elegans epithelia."

Quintin S, Gally C, Labouesse M

[Genesis, 2016]

The microtubule cytoskeleton has a dual contribution to cell organization. First, microtubules help displace chromosomes and provide tracks for organelle transport. Second, microtubule rigidity confers specific mechanical properties to cells, which are crucial in cilia or mechanosensory structures. Here we review the recently uncovered organization and functions of non-centrosomal microtubules in C. elegans epithelia, focusing on how they contribute to nuclear positioning and protein transport. In addition, we describe recent data illustrating how the microtubule and actin cytoskeletons interact to achieve those functions. This article is protected by copyright. All rights reserved.

Quintin S et al. (2008) Trends Genet "Epithelial morphogenesis in embryos: asymmetries, motors and brakes."

Gally C, Labouesse M, Quintin S

[Trends Genet, 2008]

Epithelial cells play a central role in many embryonic morphogenetic processes, during which they undergo highly coordinated cell shape changes. Here, we review some common principles that have recently emerged through genetic and cellular analyses performed mainly with invertebrate genetic models, focusing on morphogenetic processes involving epithelial sheets. All available data argue that myosin II is the main motor that induces cell shape changes during morphogenesis. We discuss the control of myosin II activity during epithelial morphogenesis, as well as the recently described involvement of microtubules in this process. Finally, we examine how forces unleashed by myosin II can be measured, how embryos use specific brakes to control molecular motors and the potential input of mechano-sensation in morphogenesis.