Tina Gumienny et al. (2005) International Worm Meeting "New members of TGF signaling: extracellular regulators of the Sma/Mab pathway"

Tina Gumienny, Huang Wang, Richard Padgett, Cole Zimmerman

[International Worm Meeting, 2005]

Growth factors and their downstream signaling pathways play vital roles in many processes, from development to homeostasis, and are targeted in many cancer cell types. However, how these growth factor pathways are regulated at or before the level of the receptors on the target cell has remained largely elusive. We have used the C. elegans TGF superfamily pathway that regulates body size and male tail formation (Sma/Mab) to improve our understanding of the regulation of growth factor signaling and have focused on two interesting conserved genes. Mutations in core pathway genes, which encode the ligand dbl-1, the receptors sma-6 and daf-4, and Smads sma-2, sma-3, and sma-4, result in animals that are smaller than the wild type and have male tail ray fusions and crumpled spicules. lon-2 was identified by Brenner as a gene involved in body length regulation, as mutations in it result in animals that are longer than the wild type. lon-2 acts genetically upstream of dbl-1. We show that LON-2 is a conserved glypican that regulates DBL-1 signaling, and is expressed in the intestine to regulate DBL-1's activity in the hypodermis. The Drosophila glypican dally can partially rescue the body size defect in animals without functional lon-2. Glypicans have been shown to affect growth factor signaling, but the precise mechanism remains unclear. We propose that LON-2 is a negative regulator of DBL-1, acting as a reservoir for ligand and affecting DBL-1's interaction with or activation of its receptors. The Padgett lab also performed a screen for Sma animals and identified lesions in a gene we named sma-10 (Savage-Dunn et al, Genesis (2003) 34(4): 239-247). It acts genetically in the unique position between lon-2 and the ligand. We cloned this gene and found that it encodes a predicted extracellular protein with immunoglobulin domains and leucine rich repeats. There is one related protein in Drosophila and three in vertebrates, though there is little genetic information on these genes and nothing that yet links them to growth factor regulation. SMA-10:GFP localizes to the hypodermis where sma-10 expression is required, and where the core dbl-1 pathway acts. The Drosophila homolog rescues the sma-10(lf) Sma phenotype. Interestingly, neither lon-2 nor sma-10 affects male tail morphogenesis, suggesting cell-type specificity in their regulation of dbl-1. We propose that SMA-10 defines a new family of positive regulators that are required for TGF signaling.

Tina Gumienny et al. (2006) Development & Evolution Meeting "Extracellular Regulators of the TGF&beta Sma/Mab pathway"

Cole Zimmerman, Huang Wang, Tina Gumienny, Richard Padgett

[Development & Evolution Meeting, 2006]

Growth factors and their downstream signaling pathways play vital roles in many processes from development to homeostasis, and are targeted in many cancer cell types. However, how these growth factor pathways are regulated at or before the level of the receptors on the target cell has remained largely elusive. Using the C. elegans Sma/Mab pathway, defined by TGFβ superfamily member DBL-1, we have investigated the regulation of growth factor signaling and have focused on two interesting conserved genes. Mutations in core pathway genes result in animals that are smaller than the wild type and have male tail ray fusions and crumpled spicules. lon-2 was identified by Brenner as a gene involved in body length regulation, as mutations in it result in animals that are longer than the wild type. lon-2 acts genetically upstream of the ligand dbl-1. We show that LON-2 is a conserved glypican, as the Drosophila glypican dally can partially rescue the body size defect in animals without a functional lon-2 gene. Glypicans have been shown to affect growth factor signaling, but the precise mechanism remains unclear. We propose that LON-2 is a negative regulator of DBL-1, acting as a reservoir for ligand and affecting DBL-1's interaction with or activation of its receptors. The Padgett lab also performed a screen for Sma animals and identified lesions in a gene we named sma-10 (Savage-Dunn et al, Genesis (2003) 34(4): 239-247). It acts genetically in the unique position between lon-2 and dbl-1. We cloned this gene and found that it encodes a predicted extracellular protein with immunoglobulin domains and leucine rich repeats. There is one related protein in Drosophila and three in vertebrates, though there is little genetic information on these genes and nothing that directly links them to growth factor regulation. SMA-10:GFP localizes to the hypodermis where sma-10 expression is required, and where the core dbl-1 pathway acts. The Drosophila homolog rescues the sma-10(lf) Sma phenotype. Interestingly, neither lon-2 nor sma-10 affects male tail morphogenesis, suggesting cell-type specificity in their regulation of this pathway. We propose that SMA-10 defines a new family of positive regulators that are required for TGFβ signaling.

Tina L Gumienny et al. (2004) East Coast Worm Meeting "LON-2 is a glypican heparan sulfate proteoglycan that regulates the Sma/Mab TGF-beta pathway in C. elegans"

Huang Wang, Tina L Gumienny, Richard W Padgett

[East Coast Worm Meeting, 2004]

The TGF-beta growth factor superfamily and its downstream core signaling components are found in all multicellular animal phyla studied, from nematodes to mammals, and are responsible for controlling several different developmental processes. They regulate pathways controlling cell cycle progression, apoptosis and immune functions, and suppress some types of cell growth. Because of such roles, they are frequently mutated in metastases. While the core components of this pathway have been identified and well-analyzed, the extracellular regulators of this pathway are not as well understood. Our lab focuses on the TGF-beta pathway that regulates body size and male tail ray development (the Sma/Mab pathway). Mutations in this pathway that prevent signal transduction result in small animals. lon-2(lf) mutant animals, however, are longer than the wild type. lon-2 acts genetically upstream of the TGF-beta superfamily member dbl-1 . The protein contains the sequence motifs characteristic of glypican heparan sulfate proteoglycans, including an N-terminal signal sequence, 12 of the 14 conserved cysteines, a glycosaminoglycan attachment site, and a glycosyl phosphatidylinositol (GPI) linkage site. Unique to LON-2, though, is an RGD integrin-binding motif. It is closest in sequence homology to Drosophila dally (division abnormally delayed ) and mammalian Glypican-3, which have been shown to modulate TGF-beta superfamily member signaling. dally rescues the Lon phenotype of lon-2(lf) animals. The lon-2 promoter driving GFP expresses in the intestine, as do the TGF-beta receptor genes sma-6 and daf-4 and the Smad gene sma-3 (though none is required in the intestine). However, a rescuing LON-2 translational fusion with GFP fluoresces in neurons in the head and ventral nerve cord, similar to dbl-1 . Based on genetic, sequence, and functional analyses, we propose that LON-2 acts at the extracellular surface to negatively regulate DBL-1 signaling.

McReynolds, Melanie et al. (2017) International Worm Meeting "Metabolic carbon tracing reveals that disrupted glycolysis arises from a loss of C. elegans salvage NAD+ synthesis."

Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

As a key link connecting regulatory and bioenergetics process, NAD+ has emerged as a central metabolic co-factor playing a critical role in regulating cellular metabolism and energy homeostasis. Successfully recycling NAM, released from NAD+-consuming reactions, back to NAD+ is a major challenge for maintaining NAD+ metabolism and homeostasis. The first step in C. elegans' salvage NAD+ synthesis involves the nicotinamidase, pnc-1 (T L Vrablik, Huang, Lange, & Hanna-Rose, 2009). Via global metabolic profiling we previously linked loss of salvage NAD+ synthesis, via loss of pnc-1 activity, to disruptions in glycolysis and a subsequent reproductive developmental delay (Wang et al., 2015). However, the citric acid cycle was not perturbed and functions of the mitochondria were intact in pnc-1 mutants (Wang et al., 2015). This observation suggested that loss of salvage NAD+ biosynthesis affects the cytoplasm specifically. To investigate this model, we decided to directly test the effects of loss of pnc-1 on glycolytic and citric acid cycle flux via application of metabolic isotopic carbon tracing tools. After a short four-hour exposure with universally labeled glucose (13C6-Glucose), we were able to detect an isotopically labeled glucose pool in both wild-type animals and pnc-1 mutants. Metabolic carbon tracing confirmed that glycolysis is impeded at the NAD+-dependent step in pnc-1 mutants. Although glycolysis is compromised, we observed no change in the isotopic flow of label from pyruvate to lactate in pnc-1 mutants. Additionally, we observed an increase of isotopic label from pyruvate entering the TCA cycle in these mutants. Although trehalose steady state levels are increased in pnc-1 mutants, we did not observe increased shunting of glucose towards trehalose. This data bolsters the model that loss of NAD+ salvage biosynthesis preferentially affects cytoplasmic functions. Moreover, our work supports stable isotopic labeling as a robust technique to track the passage of metabolites through metabolic pathways in C. elegans.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Tina Gumienny et al. (2007) International Worm Meeting "C. elegans SMA-10 is an evolutionarily conserved protein required for TGFb signaling."

Richard Padgett, Huang Wang, Jeffrey Wrana, Tina Gumienny, Cole Zimmerman, Lesley MacNeil

[International Worm Meeting, 2007]

Members of the transforming growth factor <font face=symbol>b</font> (TGF<font face=symbol>b</font>) superfamily of secreted molecules have major roles in cell growth and differentiation. Because of the role known pathway members play in disease, tumor suppression and metastasis, discovering what regulates them is critical, and may provide targets for pharmacological intervention. TGF<font face=symbol>b</font> signaling pathways are absolutely conserved, and in C. elegans a DBL-1 TGF<font face=symbol>b</font> signaling pathway is required for determining body size. Through forward genetic screens for small (Sma) animals, we isolated several alleles of a gene we called sma-10. We cloned it and showed that it is a conserved member of the LRIG (leucine-rich repeats and immunoglobulin-like domains protein) family. Recent studies of LRIG1 and LRIG2 reveal an emerging role for these molecules in cell proliferation and tumorigenesis, but their mechanism of action remains largely unknown. We show that SMA-10, like the core TGF<font face=symbol>b</font> signaling pathway components, is required in the hypodermis for body size regulation. A Drosophila homolog can rescue the phenotype of sma-10(lf) animals. SMA-10 directly binds DBL-1''s receptors, SMA-6 and DAF-4. Our studies define the role of SMA-10 and elucidate a potential conserved mechanism of LRIGs in growth factor signal transduction.

Lisa L Maduzia et al. (2001) International C. elegans Meeting "sma-11 encodes an EMK family serine threonine kinase that modulates TGF-beta signaling"

Richard W Padgett, Lisa L Maduzia, Andrew F Roberts, Stephen Cohen, Huang Wang

[International C. elegans Meeting, 2001]

Members of the Transforming Growth Factor beta (TGF-beta) superfamily affect many processes including cellular proliferation, differentiation, development and death. Mutations in several pathway components in humans result in various cancers including colon, pancreatic and breast carcinomas, as well as cause defects in bone and heart formation. In order to better understand the effects of TGF-beta on these processes, we are studying the signaling mechanisms of the Sma/Mab TGF-beta-like pathway in C. elegans . The Sma/Mab pathway is comprised of the ligand, dbl-1 , two receptor serine/threonine kinases, sma-6 and daf-4 , and the downstream effector Smads, sma-2 , -3 , and -4 . Mutations in any of these components result in animals that are small in body size (Sma) with defects in male tail (Mab) morphogenesis. We have performed several screens based on body size to isolate novel Sma/Mab pathway components. Of the many small mutants recovered from our screens, we have focused on sma-11 (see abstract by Sengupta lab on sns-8, which is allelic to sma-11 ). Although much of the general TGF-beta signaling cascade has been revealed, many issues still remain unresolved. We are particularly interested in understanding how Smad activity is regulated. We have found that sma-11 encodes a serine threonine kinase with homology to the EMK (ELKL motif kinase) family of kinases. These kinases have been shown to phosphorylate microtubule associated proteins, thereby affecting microtubule (MT) stability, and are essential for cell and embryonic polarity (Drewes 1997). Recently, it has been demonstrated that mammalian Smads are able to physically interact with beta tubulin (Dong 2000), suggesting that microtubules might play a role in retaining Smads in the cytoplasm. The molecules involved in regulating the interaction of Smads with MTs are unknown, and no genetic evidence currently exists to gauge the importance of this interaction to TGF-beta signaling. We have performed immunoprecipitation experiments in Drosophila S2 cells and find that SMA-2 binds beta tubulin. Our data suggest that this interaction is lost when the TGF-beta pathway is stimulated. We hypothesize that the SMA-11 kinase may be the trigger that allows the Smads to dissociate from MTs thereby allowing them to transmit a robust TGF-beta signal. We are currently testing this model to determine whether SMA-11 stimulates the release of SMA-2 from the microtubules. Given the strong mutant phenotype of sma-11 , it is likely that association and release of Smads from MTs may be a fundamental requirement for TGF-beta signaling. Therefore, we are further investigating the role sma-11 plays in regulating Smad activity and TGF-beta signaling.

Tina L Gumienny et al. (2002) East Coast Worm Meeting "Characterization of lon-2 and its suppressors"

Andrew Roberts, Tina L Gumienny, Lisa Maduzia, Cole Zimmerman, Huang Wang, Richard W Padgett

[East Coast Worm Meeting, 2002]

The TGF growth factor superfamily and its downstream core signaling components are found in all multicellular animal phyla studied, from nematodes to mammals, and are responsible for controlling several different developmental processes. Members of the superfamily are important for establishing the basic body plan. They regulate cell cycle progression, apoptosis and immune functions, and suppress some types of cell growth. Because of such roles, they are frequently mutated in metastases. C. elegans has three characterized TGF genes, dbl-1, daf-7, unc-129, and each has a different signaling pathway, controlling body size/male tail development, dauer formation, and pioneer axon guidance, respectively. We focus on the dbl-1 pathway. lon-2 acts genetically upstream of the ligand dbl-1. Based on genetic and sequence analysis, we predict that LON-2 acts at the extracellular surface to negatively regulate DBL-1 signaling. GFP expressed from the lon-2 promoter is strongly expressed in the intestine, as are the TGF receptors sma-6 and daf-4 . To identify novel components of the TGF signaling pathway, we screened for mutations that suppress the lon-2(lf) phenotype (see also abstract by Zimmerman et al.) Suppressors include 1) mutants of known TGF pathway genes, 2) the C. elegans homolog of Drosophila Schnurri, a transcription factor in the pathway, and 3) several novel alleles. These novel suppressors are currently being characterized and cloned (see also abstract by Maduzia et al.).

Kendall Wu et al. (2004) West Coast Worm Meeting "Identification of Genetic Biomarkers and Regulators of Aging in C. elegans"

James Lund, Min Jiang, John Wang, Yueyi Liu, Stuart Kim, Kendall Wu

[West Coast Worm Meeting, 2004]

Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the age-1 and daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.