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.

Zhang, Yan-Ping et al. (2017) International Worm Meeting "Using the CRISPR/Cas9 technology to dissect the spatial-temporal expression patterns and the functions of DAF-16 isoforms."

Zhang, Yan-Ping, Zhai, Chao, Zhang, Wen-Hong, Dong, Meng-Qiu

[International Worm Meeting, 2017]

DAF-16 is a pivotal transcription factor that transmits most, if not all, of the signaling from DAF-2 (the worm insulin/IGF-1 receptor) to regulate development, stress resistence, and aging in C. elegans. Previous studies analyzing the functions of different daf-16 isoforms produced contradictory results (Kwon ES, Nature 2010; Chen AT, Genetics 2015). In this study, we dissected the functions and expression patterns of individual daf-16 isoforms using the CRISPR/Cas9 technology, which became available in recent years. We knocked in a GFP coding sequence before the stop codon of all daf-16 isoforms at the endogenous locus and then introduced frameshifts to delete individual isoforms. We found that isoforms a, b, and d/f are the major isoforms translated into proteins, because deleting all three (d and f are treated as one) completely abolished the DAF-16::GFP signal. The expression patterns are: DAF-16a::GFP: neurons, intestine, hypodermis, muscles; from 3-fold embryos to adults DAF-16b::GFP: neurons; from bean-stage embryos to adults DAF-16d/f::GFP: germ cells, oocytes, occasionally in one or a few intestinal cells; from L4 to adults The differences in the expression were accompanied by differences in functions. Knocking out daf-16a but not daf-16b or daf-16d/f fully repressed the constitutive dauer formation phenotype induced by daf-2(e1370), indicating that the widely expressed somatic isoform daf-16a is required for dauer formation, whereas the neuronal daf-16b and germline-limited daf-16d/f are not. For thermo tolerance conferred by daf-2(e1370), at least two daf-16 isoforms were required, although a seemed to be more important than b and d/f. In contrast, the three daf-16 isoforms must all be expressed to fully support the longevity phenotype of daf-2(e1370). Ranked by how well each isoform supports daf-2(e1370) longevity, we had d/f > a > b, which was determined by leaving only one daf-16 isoform to be expressed and deleting the other two. Intriguingly, when ranked by how much the daf-2 longevity is suppressed by deleting only one daf-16 isoform, we had a > d/f > b. We also assessed the importance of the three daf-16 isoforms in lifespan extension caused by daf-2a knock-out and obtained the same results (the protein product of daf-2a was detected only in the germline, see abstract by Wen-Hong Zhang et al.). The above results revealed new insights: first and most unexpected, DAF-2/DAF-16 signaling is present in the germline and is important for lifespan regulation; besides, there are complex genetic interactions between daf-16 isoforms and cross-tissue communications between parallel channels of insulin signaling.

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.

Chamberlin HM et al. (1991) International C. elegans Meeting "INDUCTION DURING MALE SPICULE DEVELOPMENT"

Chamberlin HM, Sternberg PW

[International C. elegans Meeting, 1991]

We have begun to characterize cell interactions that specify cell fates in the male-specific blast cell, B. The B cell generates the spicules in the male tail. As shown by Sulston and White [Dev Biol 78, 577 (1980)], the fates of two pairs of bipotent cells, alpha/ (B.a( l/r)aa) and gamma/delta (B.a(l/r)pp), depend on their relative positions. Thus they must respond to spatial cues. Our cell ablation experiments indicate that other male-specific blast cells, F and U, provide at least part of such spatial cues. In a wild type male, F daughters are positioned immediately anterior to the gamma/delta pair ( next to r), and U daughters are anterior to alpha/ (next to a). Ablation of both F and U results in three B lineage defects: (l) transformation of alpha to , (2) abnormal truncated gamma lineages, and (3) abnormalities in B.a(l/r)ap. In single ablations of either F or U, the B lineage is either wild type or occasionally abnormal, with truncated gamma lineages (for F-), or alpha to transformation (for U- ). Variation in the lineages of F-U- ablated animals suggests that we have not completely eliminated the inductive signal(s). However, we have clearly identified candidates for inducing cells in the alpha/ and gamma/delta equivalence groups. Genes identified for their role in vulval induction are also involved in induction of the alpha and gamma fates. The lineage defects in mutant males suggest that, as in the vulva, there are two classes of mutations which give two opposite phenotypes. Vulvaless class mutations lin-3, let-23, lin-45, and let- 60(dn) result in transformation of alpha to , truncated gamma lineages, and abnormalities in B.a(l/r)ap. Thus, the Vulvaless mutations mimic F-U- ablations in the male B lineage, in the same way that they mimic anchor cell ablation in the hermaphrodite vulva. In contrast, Multivulva class mutations lin-l and let-60(gf) result in rare to alpha transformations, and extra divisions in delta.

Tan, Pei Yi et al. (2013) International Worm Meeting "SPV-1, a RhoGAP and F-BAR domain protein, regulates spermatheca contractility."

Tan, Pei Yi, Zaidel-Bar, Ronen

[International Worm Meeting, 2013]

C. elegans ovulation involves the passage of an oocyte through an epithelial "pouch" termed the spermatheca, where the oocyte is fertilized and subsequently propelled into the uterus. Actomyosin contractility is known to provide the force to squeeze the spermatheca, but how it is regulated in a cyclical manner is not known. We identified SPV-1, an F-BAR and RhoGAP protein, as a negative regulator of spermatheca contractility. Loss of SPV-1 resulted in over-constriction of the spermatheca, leading to premature embryo exit. Conversely, overexpression of SPV-1 transgene extended oocyte retention in the spermatheca. Expression of deletion constructs in the mutant background point to an essential role for the RhoGAP domain in regulation of contractility and for the F-BAR domain in localization of SPV-1. Full length SPV-1::GFP fusion protein revealed exclusive spermatheca expression, with transient localization to the apical cell membrane. We observed detachment of SPV-1::GFP from the membrane upon spermatheca cell stretching by an incoming oocyte, and speculated this to be due to the membrane curvature-sensing ability of its F-BAR domain. We tested this idea by expressing the F-BAR domain of SPV-1 fused to GFP in HeLa cells. In adherent cells the F-BAR domain was cytoplasmic. Upon trypsinization, when cells round up and form membrane folds, we observed translocation of the F-BAR domain to the plasma membrane. Taken together, our findings support a model in which SPV-1 localization to the apical membrane of the spermatheca is mediated by its F-BAR domain in a curvature-dependent manner. When localized to the membrane it inhibits contractility by inactivating RHO-1 through its RhoGAP domain. Oocyte entry stretches the spermatheca, straightens out the curvature and thus leads to detachment of SPV-1. We hypothesize that cytoplasmic SPV-1 looses its RhoGAP activity, possibly due to autoinhibition, permitting the level of active RHO-1 to rise beyond the threshold needed to induce contraction. In summary, we have identified an important regulator of spermatheca function and suggest a mechanism for feedback between membrane tension and actomyosin contractility that may be operating in other contractile systems.