Nahar, Saifun et al. (2021) International Worm Meeting "Glucose-induced developmental delay is modulated by insulin signaling in C. elegans"

Padilla, Pamela, Song, Luhua, Ruiz, Manuel, Nahar, Saifun, Robledo, Jose

[International Worm Meeting, 2021]

A significant amount of research has shown that type 1 diabetes in children causes a delay in puberty. However, it is unclear how hyperglycemia contributes to this delay in puberty. Hyperglycemia, which is associated with diabetes, refers to elevated blood glucose levels. To model hyperglycemia at a molecular level, C. elegans are fed a glucose-supplemented diet and the physiological and molecular impacts are studied. We found that a glucose-supplemented diet induces a developmental delay in comparison to the control food (OP50 E. coli diet). The glucose induced developmental delay was also observed in animals fed the deltaPTS E. coli OP50 bacteria strain, which lacks a glucose transporter, indicating the phenotype is not due to metabolites produced by OP50. Developmental delay can be induced by environmental stress or genetic mutations. For example, mutations that impact ceramide biosynthesis, electron transport chain activity, ubiquinone biosynthesis, or insulin signaling (hyl-2(tm2031), isp-1(qm150), clk-1(e2519) and daf-2(e1370), respectively) lead to developmental delay. We tested if a glucose diet prolonged developmental progression in these mutants. A glucose-supplemented diet further exacerbates the developmental delay of the hyl-2(tm2031), isp-1(qm150) and clk-1(e2519) mutants but did not induce a developmental delay in the daf-2(e1370) animal. The resistance to glucose-induced developmental delay observed in the daf-2(e1370) animal is suppressed by daf-16(mu86). To further understand how glucose impacts insulin signaling and developmental progression we are using RNA sequencing and genetic analysis to identify the DAF-16 regulated genes that have a role in developmental progression. This could provide insight into how hyperglycemia and altered insulin signaling is linked to delayed puberty in children with type 1 diabetes.

Robledo, Jose et al. (2021) International Worm Meeting "Identification of transcriptional regulators impacted by a glucose-supplemented diet in C. elegans"

Burks, David, Robledo, Jose, Padilla, Pamela, Song, Luhua, Azad, Rajeev, Leguizamo, Isai

[International Worm Meeting, 2021]

A high glycemic diet (i.e. high sugar diet) is among the main factors contributing to severe and detrimental human health issues such as type 2 diabetes, cardiovascular diseases, and metabolic abnormalities. Underlying mechanisms involved in gene expression regulation and metabolism as a result of a high sugar diet continue to remain poorly understood. In our study, we use the genetic model system Caenorhabditis elegans to further investigate the impact a glucose-supplemented diet has on gene expression changes/regulation. Previously, we showed that a glucose-supplemented diet increases lipid composition, sensitivity to stress responses, and significantly alters the transcriptome profile (RNA-sequencing studies). We observed differential gene expression chromosomal maps and identified various clusters majorly found in Chromosome IV/V. Given that a link exists between chromosomal clustering and transcriptional regulation we further sought to identify gene regulators. The transcriptomic analysis revealed 140 gene expression regulators among the 2,370 differentially regulated genes as a result of a glucose-supplemented diet. Furthermore, we identified the acs-2 gene is a glucose-responsive gene, as shown by RNA-sequencing analysis, the acs-2::GFP reporter strain, and quantitative RT-PCR. Here, we developed a targeted RNA interference (RNAi) screen to identify the transcriptional regulators that respond to a glucose diet and impact the expression of acs-2::GFP. From our screen, we identified 20 regulators that significantly impact the expression of acs-2 transcript in a glucose diet dependent manner; 18 regulators increase and 2 regulators decrease acs-2::GFP expression. We also identified 13 gene regulators that increase acs-2::GFP expression in standard RNAi diet. Several of the regulators identified also show homology to human gene regulators suggesting conserved gene expression regulators between C. elegans and Humans. Together, these studies add to our understanding of the transcriptional and molecular changes that occur in response to a glucose-supplemented diet.

King, Skylar D. et al. (2017) International Worm Meeting "The CISD-1 iron-sulfur protein impacts apoptosis in the germline of C. elegans."

Song, Luhua, Mittler, Ron, King, Skylar D., Padilla, Pamela A., Gray, Chipo F.

[International Worm Meeting, 2017]

The recently discovered CISD proteins (mitoNEET/CISD1, NAF-1/CISD2, CISD3) belong to a class of iron-sulfur (Fe-S) proteins that are localized to the mitochondria and/or ER and involved with several human pathologies including diabetes, neurodegeneration, and Wolfram syndrome 2. Phylogeny studies indicate that gene duplication occurred in vertebrates resulting in the distinct CISD1 and CISD2 genes. In C. elegans the cisd-1 gene has homology to the CISD1 and CISD2 genes in vertebrates and cisd-3.1 and cisd-3.2 has homology to vertebrate CISD3. In mammals the CISD proteins are thought to have a role in the regulation of iron, reactive oxygen species, autophagy and redox metabolism. The CISD1 and CISD2 protein levels accumulate in human epithelial breast cancer cells; suppression of the these proteins in cancer cells result in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and ROS in mitochondria, and activation of autophagy. Furthermore, the NAF-1/CISD2 was found to interact with BCL-2 leading to activation of apoptosis. Although these phenotypes are of interest the specific molecular function of the CISD genes is not well understood especially in the context of a whole organism. Here we used C. elegans to examine the function of cisd-1. The cisd-1(tm4993) mutant and cisd-1(RNAi) animals exhibit a reduction in fecundity and abnormal germline morphology. Using CRISPR technology we generated a cisd-1 transcriptional GFP reporter. The pcisd-1::GFP reporter exhibited GFP expression in the germline and other tissues including intestine. To test the hypothesis that cisd-1 has a functional role in apoptosis we used the programmed cell death (PCD) markers (CED-1::GFP, ACT-5::YFP) to examine PCD in the germline. The cisd-1(tm4993) mutant displayed a significant increase in apoptotic cells in the adult germline relative to controls. Knock-down of ced-3 function suppressed the increase of apoptotic cells within the cisd-1(tm4993) animal. These data suggest that cisd-1 has a role in PCD within the C. elegans germline. We are conducting additional experiments to determine the functional role cisd-1 has in C. elegans.

Hiebert T et al. (2017) MicroPubl Biol "Reduced pharyngeal pumping rates observed in tph-1 mutants using microfluidic electropharyngeogram (EPG) recordings."

Hiebert T, McCormick K, Chicas-Cruz A

[MicroPubl Biol, 2017]

In Caenorhabditis elegans, serotonin (5-HT) activates and controls pharyngeal pumping in response to food (Horvitz et al., 1982; Sze et al., 2000; Song and Avery 2012). Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping (Sze et al., 2000; Avery and Horvitz 1990; Song and Avery 2012). We used a microfluidic electropharyngeogram (EPG) recording platform (NemaMetrix) and associated software (NemAnalysis) to measure pharyngeal pumping in C. elegans tph-1 mutants in the presence of bacterial food (100 mg/ml E. coli OP50 in M9 buffer), following a 2-hr fasting period. Prior research has shown that a fasting period (e.g., 2-4-hr) induces elevated feeding rates for worms upon re-introduction to bacterial food (Lemieux and Ashrafi 2015). We chose to measure pharyngeal pumping during this elevated feeding phase due to our hypothesis that tph-1 animals would exhibit lower pumping rates than control worms. Pumping was recorded for 2-minute durations over three independent trials (total N2 n = 90; tph-1 n = 92). C. elegans tph-1 mutants exhibited significantly lower pharyngeal pumping rates than N2 control animals (A, N2 = 3.46 0.19 Hz; tph-1 = 0.89 0.10 Hz; mean SEM; *** p < 0.0001, 2-tailed students t-test). Pumping frequency data were pooled in A; see B for a comparison of each experimental trial.

Taoka M et al. (1994) J Biol Chem "Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate determination proteins."

Okuyama T, Yamakawa Y, Ozawa F, Hishinuma F, Kubota M, Taoka M, Minegishi A, Kondo H, Isobe T, Watanabe M

[J Biol Chem, 1994]

We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells.

Kelly Howell et al. (2002) East Coast Worm Meeting "Expression and localization of UNC-13 proteins in C. elegans"

Kristen Servent, Kelly Howell, Judith Lytle, Lynn Schwarting, Rebecca E Kohn

[East Coast Worm Meeting, 2002]

We are analyzing the expression patterns of different forms of UNC-13 proteins. unc-13 mutants are severely uncoordinated and are resistant to acetylcholinesterase inhibitors. The most abundant protein product expressed from this gene, UNC-13 LR, localizes to synapses (Kohn, 2000). UNC-13 interacts with other presynaptic proteins and affects vesicle priming (Richmond, 2001). While some mammalian homologues are found in the nervous system (Brose, 1995), the human homologue is expressed in kidneys (Song, 1999). The C. elegans unc-13 gene is predicted to express several different protein products including UNC-13 LR, UNC-13 LMR, and UNC-13 MR. L represents the left region of the protein, M represents the middle, and R represents the right. Mutations in the unc-13 L region of the gene alter expression of some protein products and result in uncoordinated movement. A large deletion in the unc-13 R region, which would eliminate all protein products, results in lethality (Kohn, 2000). We are developing antibodies to determine the localization of the different forms of UNC-13 proteins to explore the possibility that these proteins have different functions. Specifically, we are making antibodies that recognize the UNC-13 M and UNC-13 R regions. We are interested in determining whether all forms of the protein are found in neurons, and where proteins are localized within neurons. Knowledge of the localization patterns of the proteins will enable us to explore possible functions of these proteins.

Ping Gong et al. (2001) International C. elegans Meeting "Two-hybrid Screen for UNC-44 AO13 Interacting Proteins"

Dina Darwis, Jorge Munera, Anthony J Otsuka, Janna Lindemulder, Jerry Perschall, Brent Bill, Ping Gong

[International C. elegans Meeting, 2001]

Mutations in unc-44 result in axon outgrowth and guidance defects. The cloning of unc-44 demonstrated that it encodes a series of ankyrins, including a novel ankyrin called AO13 ankyrin [Otsuka, A. J. et al. J. Cell Biol. 129 , 1081-1092 (1995)]. AO13 ankyrin is unusual in that it is much larger than other ankyrins (6994 aa compared to 4377 aa for the largest vertebrate ankyrin, ankyrin G ). AO13 ankyrin is also unusual because the carboxyl end of the protein contains 7 potential transmembrane domains and Ser/Thr/Glu/Pro-rich (STEP) repeats. We hypothesize that UNC-44 AO13 ankyrin may be part of a signal transduction complex involved in axon guidance. To test this hypothesis, two-hybrid screens [Fields, S. and Song, O. Nature 340 , 245-247 (1989)] were performed with AO13-specific baits. Baits were constructed from a STEP repeat region (pDD3) and from the carboxyl terminus of AO13 ankyrin (pDD4 and pDD5). These baits were introduced into either yeast strain Y190 ( HIS3 and lacZ reporters) or AH109 ( HIS3, lacZ , and ADE2 reporters). These strains were transformed with R. Barstead's C. elegans cDNA clone libraries. A number of positive clones were obtained and are currently being analyzed. In addition to the two-hybrid approach, the UNC-44 ankyrins are being examined for changes in phosphorylation. Finally, dsRNAi is being used in an attempt to knock out AO13 ankyrin expression.

Semaya, Emily S. et al. (2017) International Worm Meeting "Abnormal ER morphology revealed through ultrastructural analysis of C. elegans atx-2 mutants."

Semaya, Emily S., Gnazzo, Megan M., Skop, Ahna R., Hall, David H.

[International Worm Meeting, 2017]

ATX-2, a conserved RNA binding protein, is the C. elegans ortholog of the human RNA binding protein Ataxin-2. Loss of Ataxin-2 leads to the late-onset neurodegenerative disease spinocerebellar ataxia type-2 (SCA2) and is associated with an increased risk for amyotrophic lateral sclerosis (ALS). To define the cellular role of ATX-2, we sought to determine the ultrastructure of ATX-2-depleted embryos and adults. ATX-2 is necessary for embryonic patterning in C. elegans (Kiehl et al., 2000) and recent work from the Skop and Song labs has shown cell division defects in atx-2 mutants (Stubenvoll et al., 2016; Gnazzo et al., 2016). To examine the cellular defects in the early embryo, we characterized atx-2 ts mutant animals (WM210 (atx-2(ne4297));(Gnazzo et al., 2016)) at high resolution. Transmission electron microscopy (TEM) of ultra-thin sections revealed that ATX-2-depleted animals exhibit fertilization and egg-laying defects. Both unfertilized oocytes and defective embryos are present in the uterus of ATX-2 depleted animals. Mutant embryos also exhibit defects in nuclear division, in which daughter cells are multinucleated. Ataxin-2 localizes to the endoplasmic reticulum (ER) in humans (van de Loo et al., 2009), yet the dynamics of this association during cell division are unclear. Preliminary data from the Skop lab suggests a role for ATX-2 in maintaining proper ER morphology, yet the ultrastructure was unknown. Using TEM of ultra-thin sections, we found that ATX-2 depleted embryos have fragmented rough ER, suggesting that ATX-2 is necessary for maintaining proper rough ER morphology during mitosis. Given the human phenotypes, ultrastructural studies of ER morphology in neurons and glia of ATX-2-depleted adult animals is currently underway. This research is supported by an NSF grant MCB-1158003 (Skop Lab) and an NIH grant OD010943 (Hall Lab).

Davis, Kristen et al. (2015) International Worm Meeting "The appetite control circuit in C. elegans."

You, Young-jai, Davis, Kristen

[International Worm Meeting, 2015]

Satiety quiescence is a behavioral state defined as a lack of movement or feeding following satiation. ASI is important for worms to enter satiety quiescence. We found the nutrients directly activate ASI. The nutrient rich stimulus we use in the lab is Luria broth (LB) and we are working to identify the active component by testing individual components of LB. Feeding is stimulated or suppressed depending not only on metabolic needs but also on environmental cues. To understand how feeding is controlled by environmental cues, we treated worms with nutrients mixed with noxious stimuli such as a high concentration of NaCl (4 M). 1 We found that these noxious stimuli override the ASI activation by nutrients, suggesting feeding, a potential result after sensing nutrients via ASI, can be suppressed in the presence of noxious stimuli. Furthermore, ASI suppression by noxious stimuli is entirely dependent on ASH function, a pair of neurons required for sensation of noxious stimuli. In an ASH genetic ablation background, ASI continue to respond to LB in the presence or absence of noxious stimuli. We believe there is a circuit between ASH, ASI, and an interneuron pair, AIA, which regulates ASI activation to nutrients. Our behavioral data show in an AIA genetic ablation background and an ASH genetic ablation background that satiety quiescence is enhanced when compared to concurrent controls, suggesting the signals from these neuron pairs inhibit ASI and satiety quiescence when they are intact. Recent work by others has identified a potential circuit between ASH and ASI that included three other neuron pairs RIM/RIC and ADF, although this circuit was not tested in the context of feeding or nutrient availability. 21. Chatzigeorgiou M, Bang S, Hwang SW, Schafer WR. tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature. 2013;494(7435):95-9. doi:10.1038/nature11845.2. Guo M, Wu T-H, Song Y-X, et al. Reciprocal inhibition between sensory ASH and ASI neurons modulates nociception and avoidance in Caenorhabditis elegans. Nat Commun. 2015;6:1-13. doi:10.1038/ncomms6655.

Song, Mi Hye et al. (2009) International Worm Meeting "How Does the Cell Control the Size of Microtubule Organizing Center?"

Song, Mi Hye, O'Connell, Kevin

[International Worm Meeting, 2009]

Microtubules form highly organized and polarized arrays. In proliferating cells, these arrays undergo cell cycle-dependent reorganization to establish bipolar spindles during mitosis. Microtubule rearrangements are largely under the control of the centrosome, the primary microtubule-organizing center. Centrosomes comprise a pair of centrioles surrounded by a mass of pericentriolar material (PCM), and undergo dynamic changes in both size and number during the cell cycle. While many components of the centrosome have been identified, how the cell regulates assembly of centrosomes remains unclear. Recently we identified SZY-20 (Suppressor of ZYG-1) that limits centrosome size by negatively regulating the Plk4-related kinase ZYG-1 at the centrosome (Song et al., Dev. Cell 15: 901-912 2008). We would like to understand how SZY-20 antagonizes ZYG-1 to influence centrosome size. Previously, we have shown SZY-20 contains conserved domains (SUZ and SUZ-C) found in a number of known RNA-binding proteins and that mutation of these domains disrupts both in vitro RNA-binding and the control of centrosome size. SZY-20 might regulate centrosome size through the RNA-binding role. To address this, we have sought to identify RNAs and proteins that interact with SZY-20. We have immunoprecipitated (IP) endogenous SZY-20 from wild-type embryonic extracts, and analyzed co-precipitating factors by mass spectrometry (MS) for proteins, and by high-throughput Illumina sequencing for RNAs. Using this approach, we have identified a large number of proteins that co-precipitate with SZY-20, including factors involved in RNA metabolism, protein synthesis and degradation, and nuclear-cytoplasmic transport. We are currently using RNAi and mutant alleles to determine which of these factors are required for embryonic viability, cell cycle and centrosome function. From this analysis, a subset of factors will be subject to further investigation to see if they exhibit physical and genetic interactions with SZY-20. Ultimately, we hope to identify new regulators of centrosome size and provide a detailed description of the molecular pathways involved.