Elaswad, Mohamed et al. (2021) International Worm Meeting "The role of MAP Kinase in modulating condensation of RNA binding proteins in the germ line"

Richmond, Grace, Elaswad, Mohamed, D'Amour, Ashley, Langerak, Shaughna, Busette, Neeke, Schisa, Jennifer, Wood, Megan

[International Worm Meeting, 2021]

The mitogen-activated protein kinase (MAPK) signaling pathway regulates numerous cellular processes in the C. elegansgerm line. In young hermaphrodites, di-phosphorylated MAPK is detected at high levels in late pachytene and in the proximal oocytes; however, when meiotic maturation is arrested for extended time in fog-2 females, activated MAPK is not detected. Interestingly, the activation of MAPK is inversely correlated with the assembly of large RNP granules in oocytes. In this study, we are addressing the hypothesis that MAPK inhibits the condensation of RNA-binding proteins (RBPs) into RNP granules. It is critical to understand how the assembly of RNP granules is regulated as they are hypothesized to maintain oocyte quality during extended meiotic arrest and other stress conditions. First, we used RNAi to knockdown mpk-1 in GFP::MEX-3 worms, and we detected ectopic condensation of MEX-3 into granules in oocytes. Since CAR-1 condenses into large granules in response to heat stress, we next asked if activated MAPK levels decrease in proximal oocytes after 1.5 hours at 31°C. We observed reduced levels of anti-diP-MAPK and the assembly of CAR-1 granules in oocytes. In parallel, to examine the effects of decreased mpk-1 expression on CAR-1 protein condensation, we used the temperature-sensitive allele mpk-1(ga111). At the permissive temperature, MAPK levels were high in oocytes, similar to wild-type worms, and CAR-1 was diffusely distributed in the oocyte cytoplasm. At the restrictive temperature, we observed reduced MAPK levels and increased CAR-1 condensation into granules. Wild-type worms at the restrictive temperature of 25°C had a similar phenotype as mpk-1(ga111) at the permissive temperature; therefore, the assembly of CAR-1 granules in mpk-1(ga111) is not due to mild heat stress. Current experiments are testing the effect of mpk-1(RNAi) in GFP::CAR-1 and GFP::CGH-1 worms. Our findings to date, support the hypothesis that MAPK activity inhibits the assembly of RNP granules; however, the mechanism by which MAPK may modulate the condensation of RNA binding proteins is not known. We plan to investigate if MAPK directly phosphorylates one or more RBPs, or if MAPK acts indirectly on RBP condensation, perhaps via the CCT chaperonin.

Pestrue, Chloe et al. (2021) International Worm Meeting "The CCT chaperonin selectively regulates phase transitions in the C. elegans germline"

Watkins, Brooklynne, Schisa, Jennifer, Elaswad, Mohamed, Pestrue, Chloe, Sharp, Katherine, Breton, Elizabeth

[International Worm Meeting, 2021]

When phase transitions of proteins are not properly regulated, proteins sometimes condense into aggregates that disrupt normal physiological function and lead to disease. Other times, condensation of RNA-binding proteins into ribonucleoprotein (RNP) granules is associated with homeostasis. The regulation of such phase transitions has been a topic of much inquiry but is still not completely understood. We are currently exploring the role of the Chaperone-Containing TCP1 (CCT) chaperonin in regulating RNP granule assembly in the C. elegans germline. Genetic screens performed by our lab and others initially identified several CCT subunits as promoters of PGL-1 granule assembly in embryos, and of MEX-3 granule assembly during extended meiotic arrest. In contrast, CCT inhibits stress granule and P-body assembly. Therefore, our goal was to carefully examine the role of CCT in regulating phase transitions of RNA-binding proteins in different developmental contexts. We used RNA interference (RNAi) to knockdown the expression of cct subunits in adult worms and imaged several GFP-tagged RNA-binding proteins using confocal microscopy. We find that in arrested oocytes in the fog-2 background, CCT inhibits the condensation of the P-body protein CGH-1. However, CCT does not appear to strongly regulate the condensation of the P-granule protein MEG-3. We are in the process of analyzing effects on additional RNA-binding proteins. We also find that in young, wild-type hermaphrodites CCT inhibits the condensation of the P-body proteins CAR-1 and CGH-1 in oocytes. However, CCT does not appear to affect the condensation of MEX-3, the P-granule proteins PGL-1 and MEG-3, or the stress granule proteins PAB-1 and TIAR-2. Taken together, we conclude that the CCT chaperonin selectively modulates RNA-binding proteins in the germline. Our results suggest that improper folding by CCT of one or more substrates in the germline, leads to aggregation for a subset of RNA-binding proteins. Possible models to explain our results include CAR-1 and CGH-1 as substrates directly folded by CCT, or alternatively, an indirect regulator of RNA-binding proteins may be a CCT substrate. We are currently testing whether subunits of Prefoldin, a co-chaperone of CCT, have similar roles as CCT subunits on CAR-1 and CGH-1 condensation.

Watkins, Brooklynne et al. (2021) International Worm Meeting "Characterization of stress-induced phase transitions in the C. elegans germline"

Pestrue, Chloe, Sharp, Katherine, Schisa, Jennifer, Watkins, Brooklynne, Breton, Elizabeth, Elaswad, Mohamed

[International Worm Meeting, 2021]

Many RNAs and RNA-binding proteins undergo condensation to form ribonucleoprotein (RNP) granules in the germline. The triggers and regulation of condensate dynamics are not yet well understood; however, stresses such as extended meiotic arrest can affect condensate size. During our investigation of regulators of RNA-binding protein (RBP) condensation, we observed unexpected dispersal of PGL-1 protein in control experiments. Using the DAF-16::GFP stress reporter, we determined that our imaging methods were inadvertently inducing a stress response. When worms were imaged immediately after slide preparation, DAF-16 remained cytosolic; however, extended time on agarose pads induced nuclear translocation of DAF-16. Our preliminary results indicate this imaging stress induces condensation of MEX-3 and CGH-1, dispersal of PGL-1 and GLH-1, and has no effect on TIAR-2, PAB-1, and MEG-3. In arrested oocytes, this stress results in dispersal of PGL-1, but not of MEX-3 and CGH-1. As a start to characterize the imaging stress, a time course study revealed that DAF-16 trans-locates to nuclei within 20 minutes; therefore, we conclude researchers studying condensation of RBPs need to be cautious and rigorous in their imaging methods. We are currently testing additional stress reporters to further characterize the type of stress inducing phase transitions. We have also begun investigating the question of why RNA binding proteins in arrested oocytes respond differently to stress by characterizing their properties. Our preliminary results suggest the large PGL-1 granules in arrested oocytes, but not MEX-3 or CGH-1 granules, are sensitive to hexanediol, indicating hydrophobic interactions are required for PGL-1 granule integrity. In heat stress experiments, MEX-3 condensation increases in both arrested and non-arrested oocytes. However, PGL-1 becomes dispersed in non-arrested oocytes. We are also currently performing FRAP experiments to determine the mobility of these RBPs in large RNP granules. Taken together, we conclude that heat stress and our inadvertent imaging stress differentially affect the condensation of RNA-binding proteins in the germline. The differences among RBPs correlate with the degree of their liquid-like properties based on preliminary hexanediol data. Our results also suggest that the large RNP granules in arrested oocytes may contain multiple phases, similar to what has been observed for P granules in embryos.

Ahmed M. Mohamed et al. (2007) International Worm Meeting "The Adaptor Molecule NCK-1 is a Component of the VAB-1 Eph RTK Signaling Pathway."

Jeffrey Boudreau, Ian D. Chin-Sang, Ahmed M. Mohamed

[International Worm Meeting, 2007]

VAB-1 is a C. elegans Eph receptor tyrosine kinase (RTK), and is involved in various aspects of neuronal and epidermal development. Null mutations in vab-1 result in abnormal neuroblast movements, defective ventral epidermal enclosure and various axon guidance defects [1-5]. We created animals carrying a hyper-active VAB-1 tyrosine kinase in the touch neurons by targeting the intracellular portion of VAB-1 to the plasma membrane via N-terminal myristoylation (MYR-VAB-1). The MYR-VAB-1 protein resulted in a penetrant, premature termination defect in the PLM axons that are independent of the EFN-1 ligand [3]. Using a candidate gene approach we identified nck-1 as a suppressor of MYR-VAB-1. nck-1 encodes a protein with three SH3 domains and a single SH2 domain that is highly similar to the Drosophila DOCK, and mammalian NCK2 protein. Characterization of the NCK-1 expression pattern indicates that, like VAB-1, NCK-1 is expressed in the neuroblasts of embryos, the nerve ring, ventral nerve cord and head neurons. Unlike VAB-1, NCK-1 is also expressed in epidermal cells. Analysis of nck-1 mutants revealed the presence of PLM axon defects similar to vab-1 mutants, where PLM axons overextended beyond their target region. Furthermore, NCK-1 seems to be required for proper amphid neuronal cell positioning and axon guidance. Western blots using anti-NCK-1 antibodies revealed two isoforms of NCK-1. The larger NCK-1 isoform was identified as a binding partner of VAB-1 in a yeast two hybrid screen. In a vab-1 null background the larger NCK-1 isoform levels are reduced suggesting that the larger NCK-1 isoform is less stable in the absence of VAB-1. Protein deletion analysis demonstrates that NCK-1 physically associates with VAB-1 through its SH2 domain in a VAB-1 tyrosine kinase dependent manner. Our results suggest that NCK-1 is involved in neuronal development, and is a component of the VAB-1 signaling pathway. We are currently investigating the role of NCK-1 in axon guidance, and its interaction with VAB-1. [1] Boulin et al., Current Biology 2006 16:1871-83. [2] George et al.,, Cell 1998 92:633-43. [3] Mohamed and Chin-Sang, Developmental Biology 2006 290:164-76. [4] Mork et al., Developmental Biology 2003 260:158-75. [5] Zallen et al. Development 1999 126:3679-92.

Zanetti, Michael R. et al. (2011) International Worm Meeting "Suppressors of daf-18 L1 arrest phenotype and investigating a role for DAF-18 protein phosphatase activity."

Zanetti, Michael R., Chin-Sang, Ian D., Hand, Peter E.

[International Worm Meeting, 2011]

The C. elegans Eph receptor tyrosine kinase (RTK), VAB-1, has been identified as an important player in signaling pathways controlling lifespan, axon guidance and morphogenetic movements. As such, it is important to understand how the individual components of this signaling cascade are functioning. Mutations in one of the downstream targets, daf-18(ok480), results in defective dauer formation and defective L1 arrest. DAF-18 is the homolog of the human tumor suppressor PTEN and is a negative regulator of an insulin/IGF-like pathway for longevity and dauer larva formation. In order to identify genes that function with daf-18 in L1 arrest we used two approaches to look for suppressors of the daf-18 L1 arrest phenotype. First, we used a candidate gene approach using RNAi knockdowns, which identified eleven suppressors. For suppressor genes that have mutants available, we will create double mutants with daf-18(ok480) to confirm suppression of the daf-18 L1 arrest phenotype. Secondly, we used an unbiased EMS mutagenesis screen, which identified twelve potential suppressors. These suppressors are being mapped to chromosome locations and non-complementation tests will be carried out to determine if they are novel suppressors. Previous work in our lab showed that VAB-1 and DAF-18/PTEN are substrates for each other and mutually inhibit each other [1]. We also have shown vab-1 mutants can partially suppress daf-18(ok480) L1 arrest. Human EphA3, a VAB-1 homolog, is frequently mutated in lung cancers, in particular a missense mutation in the tyrosine kinase domain EphA3(K761N), and predicted to have oncogenic properties [2]. To test gain-of-function properties in vivo we created C. elegans transgenic lines with human EphA3(K761N) and the analogous mutation in VAB-1(K859N). VAB-1(K859N) transgenic worms have a phenotype similar to hyperactive VAB-1 gain-of-function mutations where PLM axons show premature termination [3]. Based on the antagonistic interaction between VAB-1 and DAF-18 we predict that DAF-18 will dephosphorylate VAB-1(K859N) and rescue the premature termination defects. We will also test whether this regulation is conserved in human EphA3 and PTEN. We will report our findings at the meeting. [1] Brisbin et al. 2009, Dev. Cell 17, 459-469 [2] Ding et al. 2008, Nature 455, 1069-1075 [3] Mohamed and Chin-Sang. 2006, Dev. Biol 290, 164-176.

Jeffrey R Boudreau et al. (2003) International Worm Meeting "The VAB-1 Eph Receptor Tyrosine Kinase Interacts with the C. elegans Tumor Suppressor DAF-18/PTEN and May Regulate PI(3,4,5)P3 Signaling During Neuronal Development."

Jeffrey R Boudreau, Ian D Chin-Sang

[International Worm Meeting, 2003]

The C. elegans VAB-1 Eph RTK and its ephrin ligand, EFN-1 function in neurons for proper neuronal and epidermal morphogenesis. To identify downstream components of the Ephrin signaling we used the tyrosine kinase domain of VAB-1 as bait in a yeast two-hybrid screen. We isolated 4 independent cDNA clones of the daf-18 gene. The daf-18 gene encodes a homolog of the human tumor suppressor PTEN. PTEN is a dual-specificity protein phosphatase that can dephosphorylate protein substrates such as FAK and the adapter protein Shc but its physiological substrate appears to be Phosphatidylinositol 3,4,5-trisphosphate (PIP3), a lipid signaling molecule. PTEN antagonizes phosphatidylinositol-3 kinase (PI3K) the enzyme that is required to produce PIP3. The role of PTEN is evolutionarily conserved as Caenorhabditis elegans DAF-18/PTEN antagonizes the PI3 Kinase, AGE-1, in regulating metabolism, and life span. However, the DAF-18/PTEN in C. elegans probably does not act exclusively in the aging and dauer pathways, as this gene is pleiotropic and other phenotypes including defects in head and vulva morphogenesis and embryo lethality are observed. Phosphatidylinositol lipids, such as PI(4,5)P2 and PI(3,4,5)P3 mediate diverse intracellular signaling pathways to regulate the actin cytoskeleton during cell growth and movement. The VAB-1 kinase binds to the C- terminal portion and not the phosphatase region of DAF-18. Although very little is known about how PTEN is regulated, it has been proposed that the C-terminus regulates the PTEN activity possibly by phosphorylation. The VAB-1 interaction appears to be kinase dependent as a missense mutation (G912E), which disrupts kinase activity, abolished the interaction in the yeast two-hybrid assay. To understand how VAB-1 might act with DAF-18 we used a constitutively active VAB-1 kinase in the touch neurons that causes severe axon defects (See Mohamed and Chin-Sang, this meeting). We find that both age-1(mg44) and daf-2 (e1375) can partially suppress these axon defects. Our hypothesis is that VAB-1 has a negative influence on DAF-18, which leads to increased PIP3 levels, and this is compensated for in the age-1 and daf-2 mutants, which reduce PIP3 levels. Our finding that PTEN/DAF-18 physically interacts with the VAB-1 kinase may provide a model in which these two enzymes are necessary to set the PIP3 or PIP2 levels required for proper morphogenesis in C. elegans.

Jun Liu et al. (2007) International Worm Meeting "Identifying intracellular partners of the SAX-3/Robo signaling pathway and the introduction of a novel assay to study membrane protein-protein interactions."

Ian Chin-Sang, Jun Liu

[International Worm Meeting, 2007]

SAX-3 is a C. elegans neuronal receptor belonging to the conserved Roundabout (Robo) family of proteins that mediate repulsion in axon guidance decisions (Zallen et al., 1998). The proposed ligand for SAX-3 is SLT-1/Slit, and both sax-3 and slt-1 mutants exhibit axon guidance defects (Hao et al., 2001). However, the downstream signaling pathway is largely unknown, i.e., what signaling cascade does SAX-3 trigger upon binding to its ligand? The only identified downstream effector so far is UNC-34/Enabled, together with two other putative upstream regulators being VAB-8L and UNC-73/Trio (Watari-Goshima et al., 2007; Yu et al., 2002). Our lab has previously shown that SAX-3 may form co-receptor complexes with the VAB-1/Eph receptor (Ghenea et al., 2005). Considering that VAB-1 is a receptor tyrosine kinase and can potentially phosphorylate SAX-3, we modified the traditional yeast two hybrid screen by using VAB-1 kinase region as a bridge to phosphorylate the SAX-3 intracellular region. From this screen, we successfully isolated genes encoding the small isoform of nck-1 twice. nck-1 has been implicated to be involved in the VAB-1 signaling pathway for axon guidance decisions (see Mohamed and Chin-Sang this meeting). We have been conducting research on NCK-1 and its role in the SAX-3 signaling pathway and discovered that SAX-3 not only binds to the SH3 domain of NCK-1, but also binds to its SH2 domain in a VAB-1 kinase-dependent manner. We will further report our result in the meeting. There is an emerging tendency that axon guidance receptors can form co-receptor complexes for guidance decisions, however, the current available assays to study membrane protein-protein interactions are very limited. We describe a novel strategy to show receptor-receptor interaction at the membrane. In principle, we fuse membrane protein A to TEV (tobacco etch virus) protease, and fuse membrane protein B to a TEV protease recognition site followed by a GFP construct tagged with a nuclear localization signal (NLS). Upon interaction, TEV protease will be brought in close proximity to the TEV recognition site and cleave it, thereby releasing the NLS-GFP which will eventually enter the nuclei. On the other hand, if no interaction occurs, the GFP signal will remain on the membrane. We successfully used this assay to confirm the self-association of myristoylated VAB-1 intracellular region, which serves as a positive control. We are using this assay to confirm the putative VAB-1/SAX-3 co-receptor complexes, and will report our result in the meeting.

Yu, Fabian P.S. et al. (2011) International Worm Meeting "Characterization of a L1 axon guidance phenotype in the mechanosensory neurons and the role of lethal genes in axon guidance."

Yu, Fabian P.S., Mohamed, Ahmed M., Chin-Sang, Ian D.

[International Worm Meeting, 2011]

Our lab has been investigating the function of VAB-1, an Eph receptor tyrosine kinase (RTK), in neuronal development. vab-1 mutations result in abnormalities in epidermal morphogenesis, neuroblast movement, and also axon guidance [1-3]. We use the mechanosensory neurons with particular focus on the PLM to study the axon guidance roles of vab-1 and related genes. Work on axon guidance has mainly utilized the L4 or young adult stage of worms to characterize defects in axon development. Recent work in our lab has shown that the L1 stage provides a greater degree of sensitivity. At the L1 stage vab-1 null mutants display a 22% versus a 15% overgrowth defect, and for the hyperactive vab-1 tyrosine kinase strain L1 worms achieved 97% versus a reported 85% axonal undergrowth defect [3]. With this improved PLM scoring phenotype we aim to focus on 1) studying the role of lethal genes in axon guidance , and 2) studying genes known to be synthetic lethal with vab-1 null mutations. To overcome the issue of lethality we have used a tissue specific RNAi technique that is resistant to the spreading effects of RNAi [4]. The knockdown assay utilizes the rde-1 mutant background to inhibit RNAi activity in the worm. These worms are then injected with a touch neuron rde-1 specific rescuing construct via the mec-4 promoter along with an RNAi construct consisting of a dual mec-4 promoter flanking our gene of interest. We have created a number of constructs for lethal genes to study their role in axon guidance and will test whether they function in the vab-1 signalling pathway. Our preliminary study focused on arx-1 and arx-2 which code for the Arp 2/3 complex for actin nucleation. Both arx-1 and arx-2 display penetrant embryonic lethality using standard RNAi feeding or injection methods. In contrast, our neuronal specific RNAi approach bypassed the embryonic lethality and arx-1 or arx-2 RNAi displayed ~80% PLM undergrowth, suggesting that arx-1 and arx-2 are required in PLM for axon outgrowth. Thus this technique allows us to study the function of essential genes or vab-1 synthetic lethal genes in axon guidance. 1. Boulin, T., Pocock, R., and Hobert, O. (2006). Current Biology 16, 1871-1883. 2. George, S.E et al., (1998). Cell 92, 633-644. 3. Mohamed, A.M., and Chin-Sang, I.D. (2006). Dev. Biol. 290, 164-176. 4. Qadota, H et al., (2007). Gene 400, 166-173.

Mohamed, Ahmed M. et al. (2011) International Worm Meeting "Genetic and Molecular Characterization of VAB-1 Eph Receptor Signaling in Axon Guidance."

Boudreau, Jeffery, Mohamed, Ahmed M., Liu, Jun, Chin-Sang, Ian D.

[International Worm Meeting, 2011]

The vab-1 gene encodes for the only C. elegans Eph receptor tyrosine kinase (RTK), and is involved in various aspects of neuronal and epidermal development. Mutations in vab-1 result in abnormal cell migration and axon guidance [1-4]. Unfortunately, we still lack knowledge on the molecular mechanisms of how VAB-1 regulates cell migration and axon guidance in C. elegans. To identify components of VAB-1 RTK signaling, we utilized a hyper active form of VAB-1 (MYR-VAB-1) that caused penetrant axon guidance defects in the mechanosensory neurons [3], and screened for suppressors of the MYR-VAB-1 phenotype. Using a candidate gene approach, we identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1 Eph RTK. Analysis of nck-1 and wsp-1 mutants revealed PLM axon defects similar to vab-1 animals, where PLM axons over extended beyond their target region. Double mutant experiments indicate that both nck-1 and wsp-1 function in the same pathway with vab-1 in PLM axon guidance. In addition, we found that activation of the Arp2/3 complex via the VCA domain of WSP-1 induced defects similar to the MYR-VAB-1 phenotype. We also found that VAB-1, NCK-1 and WSP-1 are able to form a complex in vitro. Interestingly, protein binding experiments revealed that the actin-regulator UNC-34/Ena, known to function in axon extension, can physically interact with NCK-1. Genetic experiments to investigate the nature of this interaction suggest that unc-34 inhibits nck-1. Furthermore, genetic and biochemical data show that VAB-1 is able to disrupt the interaction between NCK-1 and UNC-34, and negatively regulate UNC-34. Taken together, our results provide a model of how VAB-1 signaling can induce axon termination. We propose that VAB-1 activation results in the recruitment of NCK-1, and disrupts the interaction between NCK-1 and UNC-34 to relieve NCK-1 inhibition. Consequently, NCK-1 is now able to form a complex with WSP-1 to activate the Arp2/3 complex and form extensive short branched filaments that inhibit growth cone filopodia formation. Simultaneously, VAB-1 activation results in the negative regulation of UNC-34 to prevent further actin filament extension. In summary, our work provides a pathway that connects the VAB-1 Eph RTK directly to the cytoskeleton, and suggests how VAB-1 regulates the actin cytoskeleton for axon termination. [1] Boulin et al., Current Biology 2006 16:1871-83. [2] George et al., Cell 1998 92:633-43. [3] Mohamed and Chin-Sang, Developmental Biology 2006 290:164-76. [4] Zallen et al., Development 1999 126:3679-92.