Soulavie, F. et al. (2019) International Worm Meeting "Robustness of the neuronal specification programs."

Soulavie, F., Bordet, G., Bertrand, V.

[International Worm Meeting, 2019]

The identity of specific neurons relies on transcriptional programs regulated by terminal transcription factors. The expression of such transcription factors is subject to expression noise that must be buffered by neurons so that their identity is acquired and maintained in an accurate manner. In C. elegans the pair of cholinergic interneurons AIY is specified by the expression of a combination of terminal transcription factors, the homeodomain LIM TTX-3 and the homeodomain CVC CEH-10, which form a heterodimer that activates terminal differentiation genes. Both terminal transcription factors are activated early in post-mitotic AIY and then automaintain their expression through binding sites present in their cis-regulatory sequences. We have observed, using CRISPR tagged versions of the endogenous TTX-3 and CEH-10, that the protein levels of these terminal transcription factors present an important variability between individuals. We are currently characterizing the origin of this variability. Furthermore, we have observed that in mutants of the Polycomb chromatin complex PRC1 the expression noise of the terminal transcription factors increases leading to a stochastic loss of AIY identity. We are currently analyzing the mechanism by which PRC1 buffers the variability in the terminal transcription factor levels.

Soulavie, F. et al. (2017) International Worm Meeting "An unexpected link between fusogen activity and intracellular lumen elongation."

Soulavie, F., Sundaram, M., Hall, D.

[International Worm Meeting, 2017]

Unicellular tubes with intracellular lumens are found in the mammalian microvasculature and in some invertebrate organs, including the C. elegans excretory (renal-like) system. Many unicellular tubes are seamless -they lack autocellular junctions along their length - and they adopt elongated or branched shapes. How cells become seamless tubes with such complex shapes is poorly understood. One proposed mechanism for seamless tube formation involves endocytosis of basal membrane followed by vesicle merging or exocytosis to form a lumen. An alternative mechanism involves cell wrapping to form a lumen lined by an autocellular junction, followed by membrane auto-fusion to remove that junction and become a seamless toroid. We provide evidence that the plasma membrane fusogen AFF-1 is involved in both mechanisms of intracellular lumen growth. The C. elegans excretory duct tube forms by wrapping and auto-fusion, and subsequently elongates more than five-fold and adopts an unusual asymmetric shape. Both auto-fusion and subsequent tube elongation depend on the EGF-Ras-ERK signaling cascade and its downstream target, aff-1. In the absence of aff-1, the duct retains an autocellular junction and has a dramatically shortened morphology, with the lumen only ~a third of its normal length. Super-resolution and electron microscopy showed that aff-1 mutants accumulate excess vesicles with apical cargoes adjacent to the main lumen, and large inclusions with highly convoluted membranes adjacent to and continuous with the basal membrane. Accumulation of the membrane-binding dye FM4-64 in aff-1 mutant duct cells suggests that the basal inclusions may correspond to a blocked endocytic intermediate. AFF-1 is an exoplasmic fusogen, and therefore could be suitably positioned to mediate dynamin-independent vesicle scission during endocytosis. Based on our observations, we propose that EGF signaling stimulates basal endocytosis in the growing duct tube, and that AFF-1 could be needed for scission of the internalized tubulovesicles, which ultimately contribute apical membrane to drive lumen growth. To further test this model, we are currently using the ZIF-1-dependent proteolysis system to remove AFF-1 from the duct cell after auto-fusion is complete, and following the subsequent progress of membrane trafficking. This work was supported by NIH grant GM58540 to M.S.

Soulavie, F. et al. (2015) International Worm Meeting "EGF/Ras signaling and the AFF-1 fusogen control seamless tube auto-fusion and shaping in the C. elegans excretory system."

Sundaram, M., Soulavie, F.

[International Worm Meeting, 2015]

Unicellular tubes with an intracellular lumen are found in the mammalian microvasculature and in some invertebrate organs, including the C. elegans excretory (renal-like) system. Many unicellular tubes are seamless -they lack autocellular junctions along their length - and they adopt elongated or branched shapes. How cells become seamless tubes with such complex shapes is poorly understood. We've shown that the C. elegans excretory duct forms a seamless tube by cell wrapping to form an autocellular junction, followed by membrane fusion to remove that junction and become a seamless toroid. The duct tube subsequently elongates more than five-fold and adopts an unusual asymmetric shape. Seamlessness and tube shape depend on the EGF-Ras-ERK signaling cascade. Surprisingly, we found that signaling promotes both seamlessness and elongated tube shape via a single downstream target, the plasma membrane fusogen AFF-1.Using transcriptional reporters for aff-1, we demonstrated that EGF-Ras-ERK signaling promotes aff-1 expression in the duct cell through a combination of relief of LIN-1/Ets-dependent repression and stimulation of LIN-1/Ets + EOR-1/BTB-Zn finger-dependent activation. Although duct auto-fusion occurs at the 1.5-fold stage of embryogenesis, aff-1 expression in the duct persists throughout much of larval development. In the absence of aff-1, the duct retains an autocellular junction and has a dramatically shortened morphology, with the lumen only ~a third of its normal length. Furthermore, lumen markers accumulate in a diffuse pattern adjacent to the main lumen, suggesting possible accumulation of a vesicular intermediate. Continuous expression of aff-1 cDNA in the duct cell rescues auto-fusion and tube morphogenesis, while late expression can rescue a part of the duct shape and lumen phenotypes independently of auto-fusion. Our results suggest a continuous role for AFF-1 in seamless tube morphogenesis, and reveal an unexpected link between fusogen activity and intracellular lumen elongation.

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.

Bonnie Kaas et al. (2008) C.elegans Neuronal Development Meeting "Regulation of Levels of UNC-13 at Synapses"

Rebecca Lawson, Rebecca Kohn, Thomas Limouze, Ryan Wennell, Bonnie Kaas

[C.elegans Neuronal Development Meeting, 2008]

Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in unc-13.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Atsushi Yamanaka et al. (2001) International C. elegans Meeting "Biochemical and functional analysis of SKP1 family in Caenorhabditis elegans"

Yasumi Ohshima, Kei-ichi Nakayama, Masayoshi Yada, Atsushi Yamanaka, Hiroyuki Imaki

[International C. elegans Meeting, 2001]

The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .

Soto, Martha et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Arp2/3-based Regulation of Junctional Dynamics During Morphogenetic Cell Movements and Polarization."

Bernadskaya, Yelena, Patel, Falshruti, Soto, Martha

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Morphogenesis requires dynamic interaction between the adherens junctions and the actin cytoskeleton. This interaction is mediated by ?-catenin, which was proposed to be a stable bridge between the junctions and F-actin at the apical regions of polarized epithelial cells. However, ?-catenin cannot bind to F-actin and its adherens junction partner, ?-catenin, simultaneously, suggesting the bridge is dynamic and complex. ?-catenin was thought to inhibit Arp2/3-based branched actin at the junctions for normal maturation of adherens junctions. Our research suggests a new positive role for Arp2/3-dependent branched actin in junctional maturation and during embryonic cell movements of C. elegans . In this study we investigate how progressive WAVE/Scar and Arp2/3-depen dent accumulation of ?-catenin at the adherens junctions contributes to the accumulation of F-actin at apical regions of epithelial cells. We find that removing Arp2/3-dependent actin nucleation disrupts junctional maturation and results in altered levels of adherens junctional proteins in epithelial tissues. In particular, there is a drop in both ?-catenin and F-actin levels at the apical intestine, and altered organization of the apical intestine. Subcellular fractionation reveals that loss of Arp2/3-dependent actin nucleation reduces the amount of ?-catenin in the membrane-associated pool. These data demonstrate an essential role for Arp2/3 to dynamically remodel F-actin to support adherens junctions and polarized F-actin during cell migration and tissue morphogenesis.

Ge Gao et al. (2004) Mid-west Worm Meeting "Loss of pan-1 causes a Peter Pan-like phenotype in C. elegans"

Ge Gao, Karen Bennett

[Mid-west Worm Meeting, 2004]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A pan-1(gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

De Fruyt, Nathan et al. (2021) International Worm Meeting "Neuropeptidergic modulation of C. elegans learning behavior"

Van Damme, Sara, Schoofs, Liliane, Watteyne, Jan, De Fruyt, Nathan, Beets, Isabel, Fadda, Melissa

[International Worm Meeting, 2021]

Neuropeptides are an evolutionarily conserved group of neuromodulators that regulate a wide range of adaptive behaviors, such as learning. Yet, unravelling the molecular and circuit mechanisms underlying this neuropeptidergic modulation is challenging due to the diversity of neuropeptide signaling pathways, and their 'wireless' extrasynaptic mode of action. Using reverse pharmacology, we have constructed a molecular map of the C. elegans neuropeptide-receptor network. Phylogenetic reconstruction of the evolutionary history of nematode neuropeptide systems across bilaterian animals revealed several nematode-specific diversifications of neuropeptide signaling in addition to evolutionarily ancient neuropeptide pathways. One of these ancient, conserved pathways is a neuropeptide Y/F (NPY/F)-like signaling system that is an important regulator of learning behavior both in Proto- and Deuterostomia. We found that NPY/F-like FLP-34 neuropeptides are required in serotonergic neurons for aversive olfactory associative learning, which is functionally similar to the role of NPY in vertebrate learning as well as to the role of NPF in invertebrate learning. NPY/F-like neuropeptides are released from serotonergic neurons and signal through the G protein-coupled receptor NPR-11 in the excitatory AIA interneurons to facilitate olfactory aversive learning. In addition, signaling through NPY/F-like receptor NPR-11 also affects learning in salt gustatory plasticity, a gustatory associative learning paradigm. NPY/F-like signaling is not the only neuropeptidergic signaling system affecting learning behavior; we discovered additional neuropeptides that appear to be important to the learning process as well, including peptides that are expressed in non-neuronal cells. Our current research focuses on unravelling the functions of such non-neuronal neuropeptide messengers in learning and other types of behavioral plasticity.