Naim, N. et al. (2019) International Worm Meeting "The tissue-specific role of NHR-49 in immunity versus longevity."

Naim, N., Gandhi, F. A., Ghazi, A.

[International Worm Meeting, 2019]

Germline-less animals are a well-established longevity model that recognizes a trade-off between lifespan and reproduction. Expression of the nuclear hormone receptor NHR-49 (functional homolog of vertebrate lipid metabolism regulator PPAR?) has been noted as essential to glp-1 mutant longevity, as it is thought to repurpose fats for reproduction in favor of a lifespan-extending lipid profile. Yet, as long-lived animals often exhibit other improved aspects of healthspan, our laboratory has seen glp-1 mutants have increased resistance to pathogen that additionally depends on NHR-49. Interestingly, NHR-49 is also needed for the immune resistance of fertile wild-type worms in this same experimental paradigm (slow-killing) which exposes worms to P. aeruginosa. This suggests the immunity benefit given to glp-1 worms by NHR-49 is not due to the resources gained from forgoing reproduction. We have identified tissues where NHR-49 acts to (1) promote longevity and (2) confer immune resistance to PA14. These will be discussed along with our data on the molecular mechanisms by which NHR-49 impacts immunity versus longevity.

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.

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.

Yde, Sarah E. et al. (2021) International Worm Meeting "Using the C. elegans zygote to study principles of actin cytoskeleton self-organization"

Munro, Edwin, Zaidel-Bar, Ronen, Ray, Shinjini, Yde, Sarah E., Kovar, David, Kadzik, Rachel S.

[International Worm Meeting, 2021]

The dynamic actin cytoskeleton is continually remodeled during organism development, assembling and disassembling functionally diverse filamentous (F-) actin networks at precisely the right time and place. A major question is how a cell can organize and maintain multiple F-actin networks with diverse architectures and dynamics from a common pool of actin and actin-binding proteins (ABPs) within a single cytoplasm. We use the C. elegans zygote to study this question in vivo, and purified C. elegans proteins to reconstitute cytoskeletal dynamics in vitro. We hypothesize that a series of self-organization mechanisms facilitates the differential recruitment and activation of ABPs that determine actin filament architectures and dynamics of different F-actin networks. A key determinant of F-actin dynamics and architecture is the length of filaments within a network. A powerful regulator of actin filament length is the ABP capping protein (CP), which binds the fast-growing barbed ends of actin filaments to prevent polymerization and depolymerization. Using both 'bulk' and single-molecule/filament assays,we have biochemically characterized the dynamics of C. elegans ceCP on actin filament barbed ends in vitro. We find that ceCP has a high affinity for actin filament barbed ends and a slow off-rate relative to the lifetime of an actin filament in a C. elegans zygote. To characterize the biological activity of ceCP, we compared this in vitro data to the dynamics of ceCP in the C. elegans zygote using a powerful in vivo single-molecule approach. We found that the lifetime of ceCP single molecules on barbed ends in a zygote is much shorter than in vitro, suggesting the presence of regulatory mechanisms that act on ceCP in the zygote. We also characterize ceCP perturbation phenotypes at both the whole-network and single-filament level, and determine how ceCP regulates the balance of actin assembly and contributes to the self-sorting of other ABPs to different F-actin networks. Upon depletion of ceCP in a zygote, assembly of some F-actin networks is increased, while others are diminished, indicating a role for ceCP in the proper distribution of actin amongst networks. Additionally, we are characterizing other ABPs using similar combined in vitro/in vivo approaches, with the ultimate goal of using the C. elegans zygote to determine the minimal components for self-organization of multiple distinct F-actin networks within a common cytoplasm.

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.

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.

Marc Van de Craen et al. (1999) International C. elegans Meeting "UNC-53 is involved in growth cone steering and co-localises with microtubule plus-ends"

Marc De Raeymaeker, Nina Cromheecke, Marleen Brunain, Marc Van de Craen, Christ Platteeuw, Joel Vandekerckhove, Walter Luyten, Peter Verhasselt, Luc Maertens, Johan Geysen, Thierry Bogaert

[International C. elegans Meeting, 1999]

Growth cone steering is a cyclic process which starts with filopodia outgrowth at the leading edge of the cell followed by the reading and integration of multiple directional signals that are translated into local stabilisation of the F-actin cytoskeleton in one of the growth cone spikes. Subsequent interaction of the F-actin cytoskeleton with microtubule plus-ends leads to permanent stabilisation of a part of the growth cone in the new direction. Finally, a new cycle of filopodia outgrowth is initiated in the new direction. UNC-53 (Steerin) is a novel nematode signal transduction molecule that integrates extracellular signals into cellular responses which determine the direction of migration of hypodermal cells, neurons, myoblasts and the excretory cell in C. elegans. Based on homology, Steerin is predicted to contain an N-terminal alpha-actinin-like F-actin binding domain and a C-terminal nucleotide-binding domain. The middle region predominantly consists of alpha helical structures comprising two proline rich helices and three coiled coils. A family of structurally and functionally related human Steerins has been cloned in a collaborative effort. Recombinant Steerin was shown to bind recombinant GST-SEM-5 and GST-GRB-2, suggesting that Steerin converts receptor tyosine signalling into directional signals. Steerin can also physically associate with the cytoskeleton via F-actin and microtubule plus-ends. Biochemical studies illustrated that Steerin can directly interact with F-actin. Steerin was shown to co-localise with microtubules with preference for the microtubule (+)-ends using monoclonal antibody post-fixation immunofluorescence and transient overexpression of GFP-Steerin fusion proteins in living mammalian cells. Overexpression of Steerin and a number of deletion mutants in mammalian cells has a positive effect on neuronal outgrowth, filopodia production, lamellipodia formation and cell motility.

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.

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.