Song, Myung Chul et al. (2015) International Worm Meeting "C09F5.1, the BRICHOS containing protein functions in stress resistance."

Han, Ching Tack, Song, Kyung Hee, Song, Myung Chul, Kim, Sung Hee

[International Worm Meeting, 2015]

C09F5.1, one of several genes expressed differentially in heat sensitive C. elegans mutants was isolated. To confirm whether C09F5.1 is involved in heat stress response, the expression pattern of C09F5.1 in heat stress condition was analyzed, showing the induction of expression of mRNA and protein by 33degC heat treatment in wild type N2. The induction of C09F5.1 protein by heat treatment delayed in heat shock transcription factor-1(HSF-1) mutants (sy441) compared to N2 implying that the HSF regulates the transcription of C09F5.1 as HSPs. C09F5.1 RNAi-treated worms became more sensitive to heat stress than control worms. These results indicate that C09F5.1 transcribed by HSF-1 may function in heat stress response. To find the putative functional domain of C09F5.1, the computational analysis of C09F5.1 protein was done. The results showed that C09F5.1 consists of an N-terminal region, a type II transmembrane domain and a BRICHOS domain. The various BRICHOS proteins are known related with familial dementia (BRI2), chondrosarcoma (Chondromodulin-1) and interstitial lung disease (SP-C). BRICHOS domain containing proteins function as molecular chaperone(Sanchez-Pulido, Devos et al. 2002). Furthermore recent studies revealed that BRICHOS domain directly interacts with Abeta42 peptide and delays amyloid-beta fibril formation(Willander, Presto et al. 2012, Hermansson, Schultz et al. 2014). When the C09F5.1 was expressed in animal cells (293T), the C09F5.1 protein directly interacted with Abeta42. In order to identify the chaperone activity of C09F5.1 on abnormal protein aggregation, we generated transgenic worms expressing each of the C09F5.1 protein with full length, BRICHOS deletion and N-terminus deletion in Abeta42 expressing worm. The transgenic worms expressing the BRICHOS domain deleted mutant showed the increased survival rate compared to worms expressing Abeta42 solely. These results suggest C09F5.1 BRICHOS containing protein has chaperonin functions under stress but not through the BRICHOS domain only. Currently we are studying the mechanism of protection from amyloid toxicity by the N-terminus of C09F5.1.

Myung Chul Song et al. (2010) East Asia Worm Meeting "A novel C. elegans gene, C09F5.1 is involved in responses to various stresses"

Ching Tack Han, Kyung Hee Song, Sung Hee Kim, Myung Chul Song

[East Asia Worm Meeting, 2010]

To find the unknown factors related to thermotolerance in C.elegans, we attempted to identify several genes differentially expressed in heat sensitive mutants isolated by mutagenesis with ethyl methane sulfate (EMS) compared to wild type worms. One of them is C09F5.1 gene, which shares no homology with any known heat-shock related proteins, and whose expression is reduced in heat sensitive mutants. The expression level of the gene is increased by heat-shock at 33degC with a peak at 30 min in wild type worms, and the interference of the gene by dsRNAi reduced thermotolerance of wild type worms. We could confirm the fact that the established C09F5.1 mutants(VC2139 and TM3903) also responded more sensitively than wild type(N2) to continuous heat-shock(33degC). Because it has been reported that stress resistant C.elegans mutant have increased longevity, we examined the life span of C09F5.1 mutants. But, they did not show difference in life span with that of wild type N2.The GFP expression under the promoter of C09F5.1 gene revealed the developmental and spatial specific expression pattern in hypodermal cells, vulva cells, seam cells, and intestinal cells. The GFP expression was intensive at L2 and L3 stage with the beginning of expression at embryo, and reduced in the young adult.The binding proteins of C09F5.1, gst-7 and nhr-57 are pulled down using C09F5.1- GST fusion protein and the interaction of gst-7 and C09F5.1 is reconfirmed by co-immunoprecipitation assay later. It is known that both gst-7 and nhr-57 are related with oxidative stress. gst-7 is the human ortholog of glutathione-requiring prostaglandin D synthase which is one of the skn-1(a key regulator regarding oxidative stress) target genes. Also, nhr-57 is known to be induced by hypoxic stress in intestine, and one of the target genes of hypoxic inducible factor-1(hif-1). Along with heat-stress, oxidative and hypertonic stresses also induced C09F5.1 expression resulting in the increased C09F5.1 mRNA level. With these data, we expect that C09F5.1 will function as relatively early regulator against multiple environment stress.

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.