Kaplan, Rebecca E.W. et al. (2013) International Worm Meeting "The small GTPase Ral signals via an Exocyst-GCK-2/MAP4K-p38-MAPKAPK cascade."

Kaplan, Rebecca E.W., Der, Channing J., Reiner, David J.

[International Worm Meeting, 2013]

We previously published that during EGF- and Notch-dependent developmental patterning of the C. elegans vulval epithelium, Ras switches between effectors Raf and RalGEF-Ral to promote mutually antagonistic 1 deg and 2 deg cell fates, respectively. Mammalian Ral signaling is poorly understood mechanistically but well validated in oncogenesis and metastasis. We therefore candidate-screened proteins loosely associated with Ral signaling. Three subunits of the Exocyst complex have been previously shown to bind Ral proteins; we found that loss of Exo84 but not Exo70 or Sec5 caused a phenotype similar to loss of Ral and blocked the activity of transgenic mutationally activated Ral. A CNH domain-containing MAP4 kinase has been shown to interact with the Exocyst by yeast two-hybrid and in human cells. We found that loss of a structurally related MAP4K, GCK-2, conferred effects similar to loss of Exo84. The MAP4K kinase domain mediates the putative Ral signal, but evidence of CNH domain MAP4K genetic complexity implies signaling duality, analogous to the aforementioned Ras signaling duality; antagonistic signals mediated by the same protein may contribute to developmental fidelity. Piecemeal studies on these vertebrate and invertebrate MAP4K proteins argue that they generally signal through the p38 MAP kinase cascade. We found that PMK-1/p38 functions in the same cascade as GCK-2/MAP4K, and demonstrated that the MLK-1 but not MTK-1 MAP3K functions similarly. We could not identify a MAP2K, perhaps due to redundancy amongst seven candidate paralogs. Thus we have identified a MAP kinase cascade probably initiated by Ral binding the Exocyst. We have additionally shown that a MAK-2/MAPKAP kinase, a well-known output of p38 kinases, functions in this GCK-2/MAP4K cascade and is expressed only in EGF-induced vulval precursors. We are currently investigating nuclear/cytoplasmic trafficking of p38 and MAPKAPK as an output of GCK-2/MAP4K cascade activity and plan to examine p38 activation in Ral-dependent cancer cell lines.

Shin, Hanna et al. (2015) International Worm Meeting "The small GTPase Ral signals via an Exo84-GCK-2/MAP4K-p38-MAPKAPK cascade to induce 2deg vulval fate.."

Reiner, David J., Der, Channing J., Kaplan, Rebecca E.W., Shin, Hanna

[International Worm Meeting, 2015]

Vulval precursor cells are patterned through graded action of EGF from the anchor cell in conjunction with LIN-12/Notch-mediated lateral signaling. Together these signals form the highly reproducible 3deg-3deg-2deg-1deg-2deg-3deg pattern that develops to form the vulval epithelial duct. A key protein in this process is LET-60/Ras, which in mammals is the most mutated oncoprotein in cancer. In presumptive 1deg cells EGFR promotes LET-60/Ras activation to induce 1deg fate via the canonical Raf-MEK-ERK MAP kinase cascade. We previously demonstrated that, in presumptive 2deg cells, LET-60/Ras switches effectors to activate the lesser known Ras effector RalGEF-Ral to induce 2deg fate in support of Notch. In general 1deg- and 2deg-promoting signals are strongly antagonistic. Thus, in equipotent cells Ras switches between Raf and RalGEF-Ral effectors to promote mutually antagonistic 1deg and 2deg cell fates, respectively. However, the Ral signaling cascade is poorly understood. We therefore screened candidate Ral effectors from the literature. Two subunits of the Exocyst complex have been previously shown to bind Ral proteins in other systems; we found that loss of Exo84 but not Sec5 caused a phenotype similar to loss of Ral and blocked the 2deg-promoting activity of transgenic mutationally activated Ral, indicating that Ral partners with Exo84 to promote 2deg fate. To move further downstream we analyzed a little known family of MAP4 kinases. In Drosophila Sec5 and Msn MAP4K, the ortholog of C. elegans MIG-15, physically interact and promote JNK signaling, but function in opposition to Ral signaling. Since Sec5-Msn did not act like a downstream Ral partner, we hypothesized that the paralogous CNH domain MAP4K subfamily, C. elegans GCK-2 and Drosophila Hppy, constitutes the real Ral effector. We found that loss of GCK-2 function conferred a defect similar to loss of Exo84, and Ral-Exo84 signals through a conserved GCK-2-p38 MAP kinase cascade. This cascade may further activate MAK-2/MAPKAP kinase. We propose that antagonistic ERK and p38 MAP kinase cascades promote opposing 1deg and 2deg fates, respectively. We further investigated MIG-15, and our data suggest that MIG-15 antagonizes LIN-12/Notch 2deg induction in the absence of EGF signal, an activity opposing the Ral-Exo84-GCK-2 EGF-dependent 2deg-promoting signal supporting LIN-12/Notch. We have discovered two functionally opposed CNH domain MAP4 kinases that function in vulval patterning. Perhaps Ral-GCK-2 and MIG-15 work in opposition to clearly demarcate 1deg and 2deg signals compartments through LIN-12/Notch regulation, thus increasing developmental fidelity.

Monahan, Kimberly B. et al. (2011) International Worm Meeting "RalGEF (RGL-1) signaling performs dual/antagonistic functions in vulval patterning."

Der, Channing J., Monahan, Kimberly B., Reiner, David J., Zand, Tanya P.

[International Worm Meeting, 2011]

The Ras small GTPase and oncoprotein that is mutationally activated in approximately 30% of all cancers. Ras and Ras-related small GTPases are involved in a myriad of cellular processes. Most studies have focused on how activated Ras utilizes two main effector signaling pathways, Raf-MEK-ERK and PI3K-AKT, in cancer initiation, development and progression. Recent studies have established a role for the non-canonical Ras effector pathway, Ral guanine nucleotide exchange factor (RalGEF) activation of-Ras-like (Ral) small GTPases, in pancreatic and other cancers. However, the mechanisms by which Ral GTPases are controlled in this pathway are still largely unknown, and how RalGEFs regulate the activation of Ral GTPases is now being explored. While there has been research focused on the GEF activity of RalGEFs, GEF- independent functions have been observed that remain poorly understood. Here, we use vulval patterning in C. elegans to study the GEF-independent functions of RalGEF (RGL-1). The canonical Ras effector in 1 deg fate induction is the LIN-45/Raf S/T kinase. However, we recently showed that the RGL-1/RalGEF-RAL-1/Ral signaling pathway functions in an antagonistic capacity relative to the Ras-Raf pathway, where Ras-RalGEF-Ral promotes 2 deg fate. Additionally, we found that RGL-1 function in the vulva is non-equivalent to that of RAL-1, suggesting RGL-1 has Ral-independent activity. ral-1-or rgl-1-directed RNAi enhanced the vulval hyper-induction of the activating Ras let-60(n1046gf) mutant, consistent with RGL-1-RAL-1 antagonism of LET-60/Ras-LIN-45/Raf. But surprisingly the three independent deletions of rgl-1 did not enhance. However, a rgl-1 deletion enhanced the let-60(gf) ectopic excretory duct cell defect, showing that the rgl-1 null mutation still antagonized Ras-Raf signaling. We hypothesize that in the vulva, RGL-1 encodes two opposing signaling activities.We used a RGL-1 GEF-dead mutant construct to rescue rgl-1 pro-1 deg but not pro-2 deg activity in a let-60(gf); rgl-1(0) background. Animals expressing the GEF-dead allele had an increase in pro-1 deg vulva formation when compared to animals expressing wild-type RGL-1, suggesting that the GEF-independent activity of RGL-1 is necessary to promote 1 deg fate. We further confirmed that this GEF-independent function is RAL-independent. Consistent with its pro-2 deg function, the ral-1 promoter drives GFP expression mainly in 2 deg cells during induction. However, we observed that the rgl-1 promoter drives GFP expression in both 1 deg and 2 deg vulva precursor cells. These results suggest that RGL-1 has dual, opposing functions in vulva cell fate patterning. A switch in RGL-1 signaling activity may contribute to robustness of vulval patterning.

Zand, Tanya P. et al. (2009) International Worm Meeting "The LET-60/Ras effectors RGL-1 (RalGEF) and LIN-45 (Raf) play antagonizing roles during vulval fate patterning."

Reiner, David J., Zand, Tanya P., Gonzalez-Perez, Vanessa, Der, Channing J.

[International Worm Meeting, 2009]

Anchor cell-dependent LIN-3/EGF signaling induces vulval precursor cells (VPCs) to assume a 2 deg -1 deg -2 deg pattern, yet current models conflict on the mechanism underlying this patterning. The "Morphogen gradient" model posits that spatially graded LIN-3/EGF signal differentially induces both 1 deg and 2 deg cell fates, while the "Sequential Induction" model argues that LIN-3 only activates LET-23/EGFRLET-60/Ras in P6.p, which in turn induces P5.p and P7.p via LIN-12/Notch to adopt the 2 deg cell fate, and thus 2 deg fate is not directly dependent on the LIN-3 signal. However, a recent study showed that the LET-60LIN-45/Raf cascade is also transiently induced in P5.p and P7.p, demonstrating that presumptive 2 deg cells receive an EGF signal with unknown consequences. We provide evidence that reconciles these two models, and consequently propose the "Morphogen-Reinforced Sequential Induction" model. Recent mammalian studies established that a pivotal second Ras effector cascade, RalGEF activation of the Ral small GTPases, cooperates with Raf to promote oncogenesis and metastasis. How RalGEFRal transduces Ras activity remains poorly defined. Unexpectedly, we found that loss of RGL-1/RalGEF or RAL-1/Ral function enhanced rather than blocked the Muv phenotype of activated let-60(gf) animals. Conversely, in the let-60(gf) background we expressed in VPCs either activated RAL-1(75L) or an effector domain mutant of activated LET-60(12V,37G) that signals primarily through RGL-1 and not LIN-45/Raf. Both constructs suppressed the let-60(gf) Muv phenotype. Taken together, these results suggest that LET-60 signals through RGL-1RAL-1 to antagonize the function of the LET-60LIN-45/Raf cascade. In epistasis experiments, rgl-1 or ral-1 RNAi enhanced the Muv phenotype of all mutants analyzed, including the downstream lin-31 transcription factor, suggesting that RGL-1RAL-1 signaling acts parallel to or downstream of LIN-45/RafMPK-1. A ral-1 promoter::gfp fusion drives GFP specifically in 2 deg but not 1 deg lineages. Furthermore, activated RAL-1(75L) enhances EGF-independent LIN-12/Notch activity, suggesting that RGL-1RAL-1 signaling is pro-2 deg . Data from other groups suggest that the LIN-45/RafMPK-1 effector pathway is quenched in presumptive 2 deg cells. We hypothesize that LET-60 signals through LIN-45/Raf in 1 deg cells, but that in 2 deg cells LET-60 effector usage is switched to RGL-1RAL-1, and thus in presumptive 2 deg cells the LIN-3/EGF signal transduced by LET-60/Ras promotes a 2 deg fate instead of a 1 deg fate. Thus, the EGF morphogen gradient can reinforce the initial patterning established by sequential induction.

Pedone, Katherine H. et al. (2009) International Worm Meeting "A conditional C. elegans selection system for Rac targets in cancer."

Reiner, David J., Peters, Eldon C., Der, Channing J., Pedone, Katherine H.

[International Worm Meeting, 2009]

Rac family small GTPases regulate actin organization, cell cycle progression, vesicular trafficking and gene expression. Growing evidence indicates that cancer cells exploit Rac activity to drive tumor growth, invasion and metastasis. We have devised a novel selection scheme in C. elegans to identify critical components of Rac effector activity for possible pharmacologic targeting in cancer. This system is based on our finding that constitutively activated CED-10/Rac1 (Q61L missense mutation) in hypodermal cells drives lethal disruption of morphogenesis. We reasoned that second-site mutations that reduce CED-10 function also would decrease CED-10-dependent lethality, creating an unambiguous selection condition for genes required by CED-10/Rac1. Furthermore, titration of CED-10(Q61L) levels to a minimal activity threshold for 100% lethality would sensitize the system to modest drops in CED-10(Q61L) output. To meet these criteria, we developed a conditional expression system for modulation of CED-10(Q61L) levels. We drove hypodermal-specific cDNA expression with a portion of the lin-26 promoter, and for conditional expression we used an aberrantly long 3''UTR sensitive to Smg system nonsense-mediated mRNA decay. Animals expressing CED-10(Q61L)::UTRNMD in the temperature-sensitive smg-1(cc546ts) background were mostly viable at 15 deg C, but were inviable when grown at restrictive temperature (24 deg C). Incremental increases in temperature resulted in progressive severity of morphogenetic defects and lethality. Control animals exhibited a ten-fold increase in GFP expression when grown at 23 deg C relative to 15 deg C. We validated the selection system by introducing mutations in known CED-10/Rac1 effectors. p21-activated kinases (Paks) are the best-characterized Rac effectors in human cells, and we found that CED-10 signals primarily through MAX-2/Pak during distal tip cell migration. At screening temperature (23 deg C), loss of MAX-2 function suppressed CED-10(Q61L)-dependent lethality to 90%. Partial suppression by MAX-2 indicates that CED-10 uses additional effectors during hypodermal morphogenesis. Loss of the Rac effector PES-7/IQGAP restored low-level viability (0.6%), while no suppression was seen with loss of the Rac effector F46F6.2/PKN or the CED-10 effector UNC-115/AbLIM. A pilot F1 clonal screen of 200 genomes yielded two suppressor alleles that confer 15-20% survival at 23 deg C. We will study novel CED-10/Rac1 signaling components identified with our screen directly in human cancer cell lines to analyze potential involvement in Rac-dependent signaling and oncogenic growth transformation.

Jun-ichi Aikawa (2001) International C. elegans Meeting "Molecular characterization of heparan sulfate/heparin N-deacetylase/N-sulfotransferase in worms"

Jun-ichi Aikawa

[International C. elegans Meeting, 2001]

Heparan sulfate binds and activates a large variety of growth factors, enzymes and extracellular matrix proteins. These interactions largely depend on the specific arrangement of sulfated moieties and uronic acid epimers within the chains. These oligosaccharide sequences are generated in a step-wise manner, initiated by the formation of a linkage tetrasaccharide which is then extended by copolymerization of alternating a1,4GlcNAc and b1,4GlcA residues. As the chains polymerize, they undergo a series of sulfation and epimerization reactions. The first set of modifications involves the removal of acetyl units from subsets of GlcNAc residues, and the addition of sulfate groups to the resulting free amino groups. These reactions are catalyzed by a family of enzymes designated as GlcNAc N-deacetylase/N-sulfotransferases (NDST), since they simultaneously. Four members of the family have been identified in vertebrates, with single orthologs present in Drosophila and C. elegans. We have revealed tissue-specific expression pattern and unique enzymatic properties of these four isozymes1,2). In fly, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens. I reconstituted cDNA for worm NDST from EST clones and 5' RACE products. Enzymatic activities will be discussed. 1) Aikawa, J. & Esko, J. D J. Biol. Chem. 274, 2690-2695 (1999) 2) Aikawa, J., Grobe, K., Tsujimoto, M. & Esko, J. D J. Biol. Chem. 276, 5876-5882 (2001)

Singh, Ramya et al. (2017) International Worm Meeting "Derlin proteins promote Notch signalling in the C. elegans germ line."

Kowalchuck, Kevin, Singh, Ramya, Sutton, Tiffany, Racher, Hilary, Hansen, Dave, Wang, Xin

[International Worm Meeting, 2017]

Notch signaling is the principal pathway that promotes cell proliferation in the C. elegans germ line. Here we report that two members of the Derlin (Degradation in the ER) family of proteins, CUP-2 and DER-2 (R151.6), promote Notch signaling. cup-2(0); der-2(0) germ lines have fewer proliferative cells than wild-type germ lines. Depleting either one of these proteins in a germ line with enhanced GLP-1/Notch signalling suppresses the size of the proliferative zone. This suppression is stronger in cup-2(0) germ lines than in der-2(0). However, depletion of CUP-2 in two different Notch-independent tumorous genetic backgrounds is unable to suppress the tumour. This suggests that CUP-2 promotes proliferation through the Notch signaling pathway specifically as opposed to suppressing proliferation in general. We further asked if CUP-2 promotes Notch signalling in other contexts, and found that lack of cup-2 enhances the loss-of-function Notch phenotype in the AC/VU decision in gonad development; therefore, CUP-2 is likely able to promote Notch signaling in general. To help determine how CUP-2 contributes to Notch signaling, we are analyzing its expression pattern. We have produced a V5::2XFLAG tagged form of CUP-2 by using the CRISPR/Cas9 system. We find that CUP-2 is expressed throughout the germ line. We are currently studying whether the CUP-2 expression pattern is altered in a variety of mutant backgrounds.

Husson, Steven J. et al. (2011) International Worm Meeting "C. elegans mutants defective in neuropeptide amidation enzymes are abnormal in egg-laying behavior."

Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen

[International Worm Meeting, 2011]

Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by egl-3 (1, 2), and a carboxypeptidase encoded by egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.

Von Stetina, Stephen E. et al. (2009) International Worm Meeting "Finding Regulators of Cadherin-Independent Epithelialization."

Mango, Susan E., Von Stetina, Stephen E.

[International Worm Meeting, 2009]

How are polarized epithelia established and maintained? This question is of critical importance, as the loss of epithelial polarity is associated with metastasis(1). There are many well-studied protein complexes that lie in specific membrane compartments with roles integral to the epithelial cell. The E-cadherin-containing adherens junction serves to link neighboring epithelial cells together while the more basal tight junction functions to separate the apical and basolateral surfaces. For some cells, E-cadherin is the major initiator of cell polarity and epithelium formation via cell-cell adhesion(2). However, recent studies have discovered E-cadherin independent polarity pathways(3-6). C. elegans offers a powerful system to study this cadherin-independent mechanism, as E-cadherin is dispensible for the initiation of epithelial polarity in nematodes(4). We study cadherin-independent epithelium formation during pharynx development. Nine pharyngeal arcade cells undergo a mesenchymal-to-epithelial transition to link the pharynx to the outer epidermis(7). Ablation of the arcade cells results in a Pharynx unattached (Pun) phenotype, in which the pharynx fails to connect to the epidermis(7). Pun animals die as they are unable to eat. Our lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished). This screen revealed that loss of the central-spindlin component ZEN-4/MKLP1 induces a Pun phenotype because the arcade cells fail to polarize(8). We are currently studying where and when ZEN-4 is needed for arcade cell polarization. We have also undertaken a structure/function analysis of this mitotic kinesin in order to elucidate its role in epithelialization. In addition, we are in the process of cloning several mutants that were isolated in the Pun mutagenesis screen. (1). J. M. Lee, S. Dedhar, R. Kalluri, E. W. Thompson, J Cell Biol 172, 973 (Mar 27, 2006). (2). L. N. Nejsum, W. J. Nelson, J Cell Biol 178, 323 (Jul 16, 2007). (3). A. F. Baas et al., Cell 116, 457 (Feb 6, 2004). (4). M. Costa et al., J Cell Biol 141, 297 (Apr 6, 1998). (5). T. J. Harris, M. Peifer, J Cell Biol 167, 135 (Oct 11, 2004). (6). W. B. Raich, C. Agbunag, J. Hardin, Curr Biol 9, 1139 (Oct 21, 1999). (7). M. F. Portereiko, S. E. Mango, Dev Biol 233, 482 (May 15, 2001). (8). M. F. Portereiko, J. Saam, S. E. Mango, Curr Biol 14, 932 (Jun 8, 2004).