Bellanger, Jean-Michel et al. (2009) International Worm Meeting "zyg-8 promotes microtubule stability in touch neurons for proper mechanotransduction."

Bellanger, Jean-Michel, Debant, Anne

[International Worm Meeting, 2009]

zyg-8 encodes a microtubule (MT)-associated protein (MAP) that is required maternally for embryonic survival and proper anaphase spindle positioning in one-cell stage embryos (Gonczy et al., 2001). Due to the lethality of zyg-8 mutant embryos, putative later functions of the gene have not been investigated to date. Using conditional gene inactivation, we showed that zyg-8 functions throughout the animal development to ensure full embryonic viability, proper morphogenesis, fertility and egg-laying. In addition, we noticed that mutant animals are lethargic and impaired in a gentle body touch assay, suggesting a defect in mechanotransduction. This process that converts light mechanical stimuli into electrical signals in the so-called touch neurons (also referred to as MT cells) requires bundles of large-diameter MTs that are unique to these cells. We therefore postulated that ZYG-8, as a potent MT stabilizer in early embryos, may promote or maintain the integrity of these structures in adults. Consistent with such a hypothesis, we established that a zyg-8 regulatory sequence drives GFP expression in touch neurons, from embryonic to adult stages. The analysis of the morphology of these neurons in mec-4::GFP;zyg-8 mutant animals revealed defects in cell body shape as well as abnormal sprouting along some processes. Low doses of the MT depolymerizing drug colchicine lead to similar defects, indicating that zyg-8 may control the morphology and process outgrowth of touch neurons by promoting MT stability. Our phenotypic analysis indicated, however, that zyg-8 loss-of-function might lead to MT unstability through a mechanism partially distinct from that of colchicine. We thus asked whether zyg-8 may contribute to the bundling or the caliber of the atypical MTs. Strikingly, ultrastructural analysis of neuronal MTs revealed that, in contrast to wild-type animals, no large-diameter MTs can be found in touch neurons of zyg-8 mutant adults. Instead, these cells are filled with bundles of MTs whose diameter is indistinguishable from that of regular MTs found in ventral cord (VC) neurons. In addition, zyg-8 mutation had no effect on the diameter of these VC MTs, indicating that zyg-8 acts selectively on touch neuron MTs, by promoting or maintaining their large-diameter. Together, our results indicate that zyg-8 (i) ensures proper cell divisions and overall development beyond the one-cell embryo stage and (ii) promotes MT stability and architecture in touch neurons that, in turn, maintain normal cell shape, process outgrowth and physiological activity of these cells.

Michel Labouesse et al. (2008) Development & Evolution Meeting "Cytoskeleton and Junction Remodelling during C. elegans Embryonic Elongation"

Hala Zahreddine, Michel Labouesse, Christelle GALLY, Huimin Zhang, Frederic Wissler, Marie Diogon

[Development & Evolution Meeting, 2008]

Elongation of C. elegans embryos provides an invaluable system to investigate several cell biological processes occurring in all epithelia, such as establishment of cell polarity, junction assembly, cell shape changes, cytoskeleton dynamics. Embryonic elongation is a rather complex morphogenetic process since it involves distinct epidermal cells and muscles. While many important players controlling this process have been identified, their integration in time and space remain poorly defined. I will outline our recent progress in two areas that contribute to elongation, the spatiotemporal regulation of myosin II activity in the epidermis, and the mechanisms involved in remodelling fibrous organelles (FOs) in response to muscle tension. To define how myosin II activity is controlled we generated several GFP probes to visualize the actomyosin cytoskeleton in vivo. We dissected the mechanism of MLC-4/rMLC activation through phosphorylation at two conserved Ser and Thr residues. We showed that MLC-4 is primarily required in lateral seam cells, and that its activity is repressed in dorsal/ventral cells through a Rho-specific RhoGAP (RGA-2). In addition, we identified the two protein kinases that phosphorylate MLC-4 during elongation and completed the characterisation of myosin II structure by identifying its essential light chain. FOs are the adhesion complexes that attach dorsal/ventral epidermal cells to the extracellular matrix and thereby connect muscles to the cuticle. They are functionally and molecularly related to hemidesmosomes, since they contain a plakin (VAB-10) and intermediate filaments. To define how FOs are assembled and how they contribute to elongation, we performed a genome-wide RNAi enhancer screen looking for clones that decrease the viability of a weak vab-10 mutant. We identified 14 genes that have human homologues with predicted roles in transcription, RNA processing, protein stability or cytoskeleton remodelling. Most of these genes interact together and thus form a functional network. Among them, PAK-1/p21-activated kinase (a novel FO component) controls intermediate filament polymerization and/or anchoring, CRT-1/calreticulin the folding of the ECM component UNC-52/perlecan, and the HECT-domain E3-ligase EEL-1 the abundance of the UNC-52 receptor myotactin. We conclude that this functional network helps maintain FO structure when submitted to external tension.

Jean-Michel Bellanger et al. (2008) European Worm Meeting "zyg-8 may interact with a Wnt pathway to regulate embryonic and larval development."

Anne Debant, Jean-Michel Bellanger, Emilie Pellegrino

[European Worm Meeting, 2008]

zyg-8 encodes a microtubule (MT)-associated protein (MAP) that is. required maternally for proper anaphase spindle positioning in one-cell. stage embryos (Gonczy et al., Dev Cell 2001). Because of the fully. penetrant embryonic lethality associated with zyg-8 mutants, putative later. functions of zyg-8 have not been investigated to date. RNAi experiments. suggest however that this gene plays developmental roles beyond the first. embryonic division (Gonczy et al., Nature 2000; Sonnichsen et al., Nature. 2005). Performing temperature-shift experiments with novel zyg-8 ts alleles. allowed us to confirm that zyg-8 is required post-maternally, for proper. embryonic and larval development. Specifically, we showed that zyg-8. functions in dividing embryos, ensuring embryonic survival and body. morphogenesis but also later, both in embryos and larvae for normal. locomotion and egg-laying behaviors.. In an attempt to uncover the signaling pathways involved in zyg-8. functions, we combined two-hybrid data with available high-throughput. interactome-transcriptome data to build a map of biologically relevant ZYG-. 8 interactors. This approach identified MIG-5 as a promising candidate to. physically and functionally interact with ZYG-8. MIG-5 is the third member. of the evolutionary conserved Dishevelled (Dsh) family of Wnt signal. transducers and controls spindle behavior in early embryos as well as. various aspects of cell fate and cell migration during larval development.. We showed that ZYG-8 does bind MIG-5 in vitro and that the interaction is. mediated by the DEP domain of MIG-5. In COS cells, we showed that GFP-DEP. is recruited to MT-associated ZYG-8, supporting the ability of this domain. to interact with ZYG-8 in vivo. Because the DEP domain of Dsh proteins is. usually involved in the transduction of Wnt signals to the so-called planar. cell polarity (PCP) pathway, we are currently testing the hypothesis that. zyg-8 and mig-5 function together in a PCP pathway to control some aspects. of C. elegans development.. To our knowledge, this interaction would represent the first example. of a direct link between a core component of the Wnt-PCP pathway and a MAP,. providing interesting insights into the mechanisms that control MT dynamics. in response to Wnt polarizing cues.

Jean-Michel Bellanger et al. (2007) International Worm Meeting "Does zyg-8 display functions after the first embryonic division?"

Nga Nguyen, Jean-Michel Bellanger, Anne Debant

[International Worm Meeting, 2007]

Microtubules (MTs) constitute an essential cytoskeletal compartment that is broadly required for cell division, cell and organelles translocations, vesicular transport and neuronal morphology for instance. Their biology is tightly controlled by trans-acting factors that bind to the lattice and regulate its intrinsic dynamic instability. Microtubule-associated proteins (MAPs) stabilize MTs whereas several destabilizers promote MTs shortening or instability. Doublecortin (DCX) is the founding member of a family of MAPs that control neuronal migrations in the developing mammalian brain. Mutations in DCX result in a severe human disease called the double-cortex syndrome, characterized by multiple cortical heterotopia. The Doublecortin-like kinases (DCLKs) display a bipartite structure, with a Ser/Thr kinase domain associated to the family-defining DCX domain. In mice, DCLK1 acts redundantly with doublecortin during neurocortical migrations but also displays unique roles in the early generation of neuroblasts, presumably by ensuring proper mitotic spindle integrity of glial progenitor cells. Some DCLK1 isoforms as well as DCLK2 are enriched in the mature nervous system, suggesting other unknown functions of these proteins in adult mice. The unique C. elegans member of this family of MAPs, ZYG-8, displays the structure of DCLKs, strongly supporting the hypothesis of an ancestral association of the DCX module with a Ser/Thr kinase domain, with later (Vertebrates) duplication/mutational loss of the kinase domain in the Doublecortin gene. ZYG-8 binds and stabilizes microtubules through its DCX domain but importantly, mutational analysis revealed that both domains are required for proper anaphase spindle behavior during the first embryonic division. Therefore, millions of years of evolution seem to have conserved the DCX/kinase association within the same protein, for the control of spindle integrity/behavior and related biological processes. The molecular mechanisms by which DCLK/ZYG-8 proteins stabilize mitotic spindles, the role of the kinase domain, the extent of their physiological functions as well as the way they are regulated remain largely unknown. We decided to investigate these questions in C. elegans, through the use of zyg-8 ts mutants to bypass the early zyg-8 requirement and the combination of two-hybrid, transcriptomic and phenotypic data to infer signaling pathways into which zyg-8 may be involved. Our results indicate that zyg-8 may function after the first embryonic division and cross-talk with pathways involved in development, morphogenesis and potentially ER stress and longevity.

Michel Labouesse et al. (2002) European Worm Meeting "One gene for two distinct plakins that provide outer and inner resistance to mechanical stress"

R Legouis, Ann M Rose, J M Bosher, B-S Hahn, A Gansmuller, Michel Labouesse, A Chisholm

[European Worm Meeting, 2002]

To identify genes required for epidermal integrity and embryonic morphogenesis, we previously performed a chromosomal deficiency screen looking for embryos that failed to elongate (Dev Dyn 210:19-32). We next focused on a promising deficiency, hDf17, and identified an embryonic lethal mutation, h1356, mapping under hDf17 that recapitulates part of its phenotype. We showed that h1356 corresponds to a putative null allele of the gene vab-10, previously defined by the viable allele e698, and identified another strong allele, ju281 which failed to complement h1356 and e698. We showed that vab-10 encodes two distinct plakins via a complex pattern of alternative splicing. Plakins are large proteins thought to bridge different cytoskeletal elements. Specifically, all VAB-10 isoforms share a common N-terminal domain with a predicted actin- binding domain. Their C-terminal domains are either similar to plectin, a protein known to bind actin and intermediate filaments, or MACF, a protein known to bind actin and microtubules (we refer to these isoforms as CePlectin and CeMACF, respectively). Sequencing of the three vab-10 alleles shows that h1356 affects the common N-terminal region, while ju281 and e698 are missense mutations affecting the unique CePlectin region. Using polyclonal antibodies against CePlectin, we found that it is a likely component of fibrous organelles, a structure found in the epidermis that provides a mechanical link between muscles and the cuticle across the epidermis. Similarly using two antisera against CeMACF we found that CeMACF forms a pattern of parallel bands in the epidermis that is interspersed with fibrous organelles.

Michel, Agnes et al. (2009) International Worm Meeting "Mechanism of X-chromosome dosage compensation: probing X-chromosome higher-order structure in C. elegans ."

Pickle, Cathy, Meyer, Barbara, Michel, Agnes

[International Worm Meeting, 2009]

Males and females differ in their number of X chromosomes, yet both require a similar level of X-chromosome products. Dosage compensation is an essential, universal, chromosome-wide regulatory process that equalizes the somatic expression of X-linked genes between males and females/hermaphrodites. A dosage compensation complex (DCC) is directed to both X chromosomes of C. elegans hermaphrodites to reduce gene expression by half. DCC binds to discrete, dispersed cis-acting elements on X. Of the many sites bound by DCC, few can serve as DCC recruitment sites (recruitment element on X, rex). We have shown that the recruiting ability is at least partially conferred by a consensus motif (MEX) that is shared among rex sites. Most sites (dependent on X, dox) fail to recruit the DCC autonomously, therefore must be bound through a different mechanism. The similarity of DCC to condensin, a conserved protein complex essential for chromosome compaction, resolution, and segregation, suggests that DCC loading along X is associated with changes in chromosome structure. Long distances separate rex from dox sites, implying that long-range interactions are important for DCC distribution. We have recovered animals that carry homozygous deletions of rex sites. These strains will be used to test the hypothesis that DCC binding to rex sites is necessary for DCC binding to dox sites via a mechanism of long-range interaction. Chromatin immunoprecipitation experiments using DCC-specific antibodies will allow us to determine whether dox sites become unoccupied in the rex deletion strains. In parallel, Chromosome Conformation Capture will be used to determine whether the rex and dox sites contained in two well-characterized 190kb regions interact at long distances. Finally, we will establish the network of interactions of all the DCC binding sites present in a large representative portion of the X chromosome, using the method of Chromosome Conformation Capture Carbon Copy. Swiss National Science Foundation PBGEA-119304 and PA00P3_124165 / 1 NIH R01-GM30702.

Jean-Michel Bellanger et al. (2002) European Worm Meeting "ZYG-8 and TAC-1 are required for proper microtubule behaviour in one- cell stage C.elegans embryos."

Pierre Goenczy, Jean-Michel Bellanger

[European Worm Meeting, 2002]

Proper spindle positioning is essential for spatial control of cell division. We have previously reported that zyg-8 is a key component directing anaphase spindle positioning in one- cell stage C. elegans embryos (Dev Cell. 2001 Sep;1(3):363-75). ZYG-8 harbors a kinase domain and a domain related to the microtubule-associated protein (MAP) Doublecortin. We have shown that ZYG-8 is a MAP that protects microtubules against depolymerizing agents, suggesting that ZYG-8 directs anaphase spindle positioning in one-cell stage embryos by promoting microtubule stability. Anaphase spindle positioning is mainly driven by pulling forces exerted on the spindle poles along astral microtubules. We probed the extent of these forces in zyg-8 mutant embryos by severing spindle microtubules with a laser beam. Surprisingly, we found the extent of pulling forces to be like in wild-type. Therefore, zyg-8 function is not required for the generation of pulling forces. Instead, these results indicate that zyg-8 is necessary for spindle elongation in response to pulling forces exerted on spindle poles during anaphase. In order to better understand how zyg-8 promotes microtubule stability, we sought to identify molecular partners of ZYG-8 using the yeast two-hybrid system. Using the N-terminal 464 aa of ZYG-8 that encompass the Doublecortin domain as bait, we screened 0.75x106 clones and identified 76 interactors corresponding to 24 different open reading frames. We have begun to assess the biological significance of these interactions by inactivating the corresponding genes using RNAi in wild-type and zyg-8 mutant animals. This approach allowed us to identify the only C. elegans counterpart of the mammalian Transforming and Acidic Coiled Coil family of proteins as a particularly interesting candidate. We name this gene tac-1. TACC genes have been initially identified in human cancer cell lines where they are either overexpressed (Hs-TACC1 and Hs-TACC3) or underexpressed (Hs-TACC2). Subsequent work in Drosophila revealed that D-TACC is required for proper spindle and astral microtubule behaviour. D-TACC is localized to spindle poles and interacts with MAPs of the Msps/XMAP215/ZYG-9 family. Interestingly, we found that tac-1 (RNAi) embryos in C. elegans have a pronuclear migration phenotype indistinguishable from that of zyg-9 mutant embryos, characterized by compromised microtubules throughout the cell cycle. Taken together, these observations suggest that TAC-1 may interact with ZYG-9, in addition to ZYG-8, in one- cell stage C. elegans embryos. We are currently testing tenets of a working model in which TAC-1 is an essential centrosomal anchor for microtubule stabilizers like ZYG-9 and ZYG-8.

Jean-Michel Bellanger et al. (2006) European Worm Meeting "Identification, by a Proteomic Approach, of the Signaling Networks Controled by UNC-73(Trio) in the Developing Nervous System of C. Elegans"

Jean-Michel Bellanger, Anne Debant

[European Worm Meeting, 2006]

Identification, by a Proteomic Approach, of the Signaling Networks Controled by UNC-73(Trio) in the Developing Nervous System of C. Elegans. Jean-Michel Bellanger and Anne Debant. The development of the embryonic nervous system, which is remarquably conserved through evolution, requires that nascent axons extend across a complex extracellular environment to reach their specific targets. Guidance cues steer the axons towards the appropriate destination and must be relayed through the growth cone to the actin cytoskeleton. Rho-GTPases play a pivotal role in conveying signals to the actin cytoskeleton, causing morphological changes in many different cell types, and there is now compelling evidence that they transduce signals from membrane receptors to the actin cytoskeleton during axon guidance. These proteins are activated by the guanine-nucleotide exchange factors (RhoGEFs), that favor the active, GTP-bound state, of their GTPase targets. Trio is a unique RhoGEF because it contains two GEF domains with different specificity and numerous signaling motifs probably involved in protein-protein interactions. Genetic studies indicate that Trio has essential and evolutionary conserved functions in cell motility and axon guidance, by controling Rho-GTPases activity on actin cytoskeleton remodeling. Compeling evidence suggest that Trio is part of multimolecular complexes that are essential for the transmission and the integration of the intracellular signals elicited by the guidance cues. However, the composition and the functional role of these complexes remain elusive.. Our goal is to contribute identifying such complexes in the context of a developing nervous system. Three main reasons lead us to consider C. elegans as the model of choice for our project. First, Trio is represented by a unique family member in the nematode, unc-73, which is very well described, both at the genetic and molecular levels, for its roles in cell migration and axon guidance. Second, due to its relative simplicity, the worm nervous system has been described at a level that is not reachable in other systems. Consequently, a wide panel of tools (both genetic, molecular and cellular) are now available for neurobiological analyses. Third, due to its short life cycle and its ability to be grown in liquid culture, large amounts of biological material can be obtained from this model organism. Therefore, we propose: i) to identify, by a proteomic approach, the UNC-73-associated proteins in vivo, directly in the developing nervous system of C. elegans embryos or young larvae; ii) to analyze, by molecular and genetic approaches, the function of theses complexes in vivo; iii) finally, to determine whether the complexes identified in the nematode are conserved in mammals and if they are involved in the same signaling and/or developmental pathways. The poster will describe our strategy and its current experimental status.

Jean-Michel Bellanger et al. (2005) International Worm Meeting "Identification, by a proteomic approach, of the signaling networks controled by UNC-73(Trio) in the developing nervous system of C. elegans."

Laetitia Chotard, Anne Debant, Jean-Michel Bellanger

[International Worm Meeting, 2005]

The development of the embryonic nervous system, which is remarquably conserved through evolution, requires that nascent axons extend across a complex extracellular environment to reach their specific targets. Guidance cues steer the axons towards the appropriate destination and must be relayed through the growth cone to the actin cytoskeleton. Rho-GTPases play a pivotal role in conveying signals to the actin cytoskeleton, causing morphological changes in many different cell types, and there is now compelling evidence that they transduce signals from membrane receptors to the actin cytoskeleton during axon guidance. These proteins are activated by the guanine-nucleotide exchange factors (RhoGEFs), that favor the active, GTP-bound state, of their GTPase targets. Trio is a unique RhoGEF because it contains two GEF domains with different specificity and numerous signaling motifs probably involved in protein-protein interactions. Genetic studies indicate that Trio has essential and evolutionary conserved functions in cell motility and axon guidance, by controling Rho-GTPases activity on actin cytoskeleton remodeling. Compeling evidence suggest that Trio is part of multimolecular complexes that are essential for the transmission and the integration of the intracellular signals elicited by the guidance cues. However, the composition and the functional role of these complexes remain elusive. Our goal is to contribute identifying such complexes in the context of a developing nervous system. Three main reasons lead us to consider C. elegans as the model of choice for our project. First, Trio is represented by a unique family member in the nematode, unc-73, which is very well described, both at the genetic and molecular levels, for its roles in cell migration and axon guidance. Second, due to its relative simplicity, the worm nervous system has been described at a level that is not reachable in other systems. Consequently, a wide panel of tools (both genetic, molecular and cellular) are now available for neurobiological analyses. Third, due to its short life cycle and its ability to be grown in liquid culture, large amounts of biological material can be obtained from this model organism. Therefore, we propose: i) to identify, by a proteomic approach, the UNC-73-associated proteins, directly in the developing nervous system of C. elegans embryos or young larvae; ii) to analyze, by molecular and genetic approaches, the function of theses complexes in vivo; iii) finally, to determine whether the complexes identified in the nematode are conserved in mammals and if they are involved in the same signaling and/or developmental pathways. The poster will describe our strategy and its current experimental status.

Jean-Michel Bellanger et al. (2003) International Worm Meeting "ZYG-9, TAC-1 and ZYG-8 cooperate to control microtubule behavior in the one-cell stage C. elegans embryo"

Jean-Michel Bellanger, Pierre Gonczy

[International Worm Meeting, 2003]

Proper spatial and temporal control of microtubule dynamics by microtubule associated proteins (MAPs) is essential for successful cell division. XMAP215 is an evolutionarily conserved MAP that plays a crucial role in stabilizing microtubules throughout the cell cycle. In C. elegans, the XMAP215-family member is encoded by zyg-9, which is required in one-cell stage embryos for a number of microtubule-dependent processes, including pronuclear migration (Matthews, L.R., et al., J Cell Biol, 1998. 141(5): p. 1159-68). zyg-8 encodes another evolutionarily conserved MAP required for proper cell division in C. elegans (Gonczy, P., et al., Dev Cell, 2001. 1(3): p. 363-75). ZYG-8 harbors a kinase domain and a Doublecortin domain that mediates microtubule stabilization. The function of zyg-8 is required in a temporally more restricted manner than that of zyg-9 since zyg-8 mutant embryos undergo normal pronuclear migration, but exhibit defective spindle positioning during anaphase. How ZYG-8 activity is regulated throughout the cell cycle and whether it may control microtubule dynamics by interacting with other MAPs remain to be elucidated. To begin addressing these questions, we sought molecular partners of ZYG-8 through a yeast two-hybrid screen. We identified the unique C. elegans member of the Transforming and Acidic Coiled-Coil (TACCs) family of proteins. In Drosophila, D-TACC localizes to spindle poles and controls microtubule behavior (Raff, J.W., Trends Cell Biol, 2002. 12(5): p. 222-5). Moreover, D-TACC physically interacts with the XMAP215 orthologue MiniSpindles, perhaps helping to recruit it to astral and spindle microtubules, thus ensuring their stabilization. In C. elegans, we found that pronuclear migration does not take place in tac-1 (RNAi) one-cell stage embryos, just like in zyg-9 mutant embryos. Consistent with the Drosophila data, we established that TAC-1 and ZYG-9 physically interact, both in vitro and in vivo. Furthermore, antibody staining and GFP-fusion protein expression studies revealed that TAC-1 mainly localizes to spindle poles during mitosis. Intriguingly, we found that the relationships between TAC-1 and ZYG-9 may rely on a mechanism that differs in part from that described in Drosophila. Indeed, we observed that tac-1 is required for ZYG-9 protein stability, and reciprocally. We are currently investigating the molecular basis of this effect, as well as the biological significance of the interaction between ZYG-8 and TAC-1.