Daniel Marston et al. (2008) Development & Evolution Meeting "MRCK Drives Apical Constriction in C. elegans Gastrulation by Activating Myosin"

Daniel Marston, Bob Goldstein

[Development & Evolution Meeting, 2008]

Directed cell migrations drive morphogenetic rearrangements during development. We are using the cell migrations of C. elegans gastrulation as a model to understand how the actin network and its associated myosin motors are regulated by intra- and intercellular signaling networks during morphogenetic movements. Gastrulation in C. elegans is a simple system that begins with two cells, the endodermal precursor cells, moving from the ventral surface of the embryo to the embryonic interior, driven at least in part by constricting their apical surfaces via actomyosin contraction. We showed that a Wnt/Frizzled signal is required for myosin activation at the apical surface of the ingressing cells and are now investigating intracellular signaling networks. Myosin motors are typically regulated through phosphorylation of the myosin regulatory light chain by several kinases including the Rho-GTPase-family regulated ROCK and MRCK, and the calcium regulated MLCKs. We have found that both MRCK and ROCK are required for gastrulation movements but that they have different roles. Depleting ROCK activity, either by RNAi to let-502 or using a temperature sensitive allele, has only a weak effect on gastrulation, and we have identified a RhoGEF and a RhoGAP with similar phenotypes. In contrast, depleting TAG-59, the C. elegans MRCK, using RNAi causes a strong, highly penetrant defect in ingression of the endoderm cells. In embryos depleted of TAG-59, cell specification appears normal, as does the apico-basal distribution of PAR proteins. However, there is a dramatic reduction in the level of phosphorylated apical myosin. The gastrulation defect is partially rescued in a mel-11 (myosin phosphatase) background, which gives further evidence for its role in myosin activation. These results suggest that, unlike other organisms, Rho/ROCK signaling is not a primary driver of apical constriction and further suggests a new role for MRCK, that of activating apical myosin and apical constriction during these morphogenetic movements.

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.

Quintin, Sophie et al. (2015) International Worm Meeting "LET-502/ ROCK acting independently of myosin-II promotes junctional protein transport or trafficking in parallel to microtubules."

Labouesse, Michel, Quintin, Sophie, Oegema, Karen, Wang, Shaohe, Gally, Christelle

[International Worm Meeting, 2015]

During C. elegans morphogenesis, the embryo elongates through epidermal cell shape changes. Two processes are required for elongation: actomyosin contractility controlled by LET-502/ROCK activity, and muscle contractions, which trigger a mechanotransduction pathway in the epidermis through hemidesmosomes (HDs). In addition, microtubules (MTs) play a poorly understood role (Priess and Hirsh, 1986). To define how MTs contribute to elongation, we first examined their organization and dynamics in elongating embryos. We find that two minus-end MT regulators, gamma-tubulin and NOCA-1, localize to adherens junctions and along HDs. To disrupt MTs, we specifically expressed the MT-severing protein SPASTIN in the epidermis. This does not strongly affect morphogenesis, unless LET-502/ROCK activity or HDs are partially compromised. Cellular and kinetic data suggest that MTs mainly act by promoting the transport of E-cadherin to adherens junctions and myotactin/LET-805 to HDs, which are critical for elongation. Importantly, let-502/ROCK activity potentiates MT transport, independently of myosin-dependent tension. To look for genes involved in MT-mediated transport and elongation, we performed two independent RNAi screens. The first screen targeted genes whose depletion phenocopied the hypo-elongated phenotype of spastin expression in let-502(lf) animals. Tubulin folding and centrosome biogenesis genes were identified, confirming that MT levels are critical when let-502 is lowered. Among traffic candidate genes, the syntaxin SYX-5 stood out as a key player acting with LET-502. Our data suggest that syx-5 could act by promoting vesicle fusion events close to adherens junctions or HDs. In the second screen, we looked for genes acting in parallel to git-1, the earliest acting factor in the mechanotransduction pathway. We identified genes of the minus-end directed MT motor dynein/dynactin, including a novel component of the complex, FARL-11. Several members of the dynein complex, including farl-11 and dlc-1, were found to localize along HDs. Interestingly, dlc-1 localization pattern depends on a critical serine residue, which needs to be unphosphorylated. Altogether, our data suggest that HDs act as a platform where MTs could be nucleated and trapped, to allow the delivery of proteins essential for elongation. Our finding that LET-502/ROCK reinforces this process independently of its role as a myosin II activator implies that it acts through a target controlling trafficking.

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 .

Chen, Chun-Hao et al. (2013) International Worm Meeting "Microtubules-Based Inhibition of RhoA and MAPK Signaling Promotes Synapse Maturation and Axon Branch Maintenance in C. elegans."

Chen, Chun-Hao, Lee, Yu-Hsien, Pan, Chun-Liang

[International Worm Meeting, 2013]

Connectivity of the neuronal circuit is refined by the dynamic regulation of synapse formation and elimination. While mechanisms that promote synapse formation had been studied in detail, those that maintain established synapses are still poorly understood. We identified two tubulin genes, mec-12/a-tubulin and mec-7/b-tubulin, as essential for synapse and axon branch maintenance. In the mec-12 and the mec-7 mutants, the PLM synapses formed normally but were progressively lost, with concomitant retraction of the PLM axon branch. The synapse and axon branch defects of the tubulin mutants were suppressed by mutations in rhgf-1, a RhoGEF with potential microtubule association. Genetic analysis suggested that the GEF activity of rhgf-1 triggered synaptic and axon branch defects and that the PDZ and the C1 domains may inhibit this GEF activity. We further demonstrated that RHGF-1 acted through the Rho (rho-1), the Rac (ced-10 and mig-2) and the Rho kinase let-502/ROCK, but independently of the canonical ROCK target MLC-4/myosin regulatory light chain. Instead, it activated a conserved p38/MAPK pathway, consisting of dlk-1/MAPKKK, mkk-4/MAPKK, and pmk-3/MAPK. DLK-1 was required and sufficient to promote PLM branch retraction cell-autonomously through sequential phosphorylation of MKK-4 and PMK-3. These MAPK components were expressed both at the synapse and in the neuronal soma. Interestingly, we found that blocking the dynein-based retrograde transport significantly reduced axon branch defects of the mec-7 mutants. We propose a model in which microtubules disassembly activates MAPK signaling through ROCK, and this MAPK activity is part of the retrograde signals that induce subsequent axon retraction upon microtubule loss. Our work had identified molecular mechanisms by which microtubules promote synaptic stability and axon branch maintenance in the neurons. This work is supported by the National Science Council, Taiwan (NSC99-2320-B-002-080 and NSC100-2320-B-002-095-MY3) and National Taiwan University (NTU-CDP-102R7810).

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.