Kumar, Anita et al. (2021) International Worm Meeting "Nucleolar size is modulated by autophagy protein LGG-1/GABARAP"

Lapierre, Louis, Kang, Taewook, Rogers, Aric, Thakurta, Tara, Larsen, Martin, Kumar, Anita

[International Worm Meeting, 2021]

Active nuclear-cytoplasmic transport of several proteins and RNAs is facilitated by nuclear import and export receptors. Exportin 1 (XPO-1/XPO1/CRM1) is one such conserved receptor that aids nuclear export of a few mRNA and over 200 proteins including pre-ribosomal subunits, transcription factors, tumor suppressors, and oncoproteins. Previously, we had shown that genetic and pharmacological inhibition of XPO-1 conferred proteostatic benefits and enhanced lifespan in nematodes by transcriptional upregulation of autophagy via the transcription factor HLH-30/TFEB. Since XPO-1 is a major export receptor, we sought to characterize the nucleo-cytoplasmic distribution of proteins upon xpo-1 silencing. Proteomic analysis of nuclear and non-nuclear (cytoplasmic) fractions of worms upon xpo-1 RNAi by TMT-MS revealed repartitioning of several proteins involved in ribosome biogenesis, translation initiation, mRNA functions, and proteostasis. We also found transcriptional downregulation of ribosomal genes and a global reduction of protein synthesis by RNA-sequencing and polysome profiling, respectively. Additionally, the size of nucleoli, the hubs of rRNA synthesis inside the nucleus, and levels of nucleolar protein fibrillarin were significantly decreased, in line with recent reports on long-lived nematodes. Probing altered nucleolar dynamics further, we uncovered RPL-11.1, a ribosomal large subunit protein that has a role in nucleolar stress response, to be repartitioned to the nucleus in xpo-1 RNAi nematodes. We found that RPL-11.1 modulates nucleolar size in an LGG-1-dependent fashion, i.e., nuclear RPL-11.1 levels and nucleolar size increase when the autophagosome protein, LGG-1/GABARAP, is silenced. Our results also indicate an interaction between RPL-11.1 and LGG-1, highlighting a novel link between two convergent hallmarks of aging, nucleolar size and proteostasis. Furthermore, silencing rpl-11.1 in nematodes expressing Alzheimer's Disease protein, AB, in muscles, significantly reduced paralysis suggesting proteostatic benefits of reduced RPL-11.1 levels. Altogether, our data uncover that autophagy protein LGG-1/GABARAP is a key modulator of nucleolar function and lifespan by surveilling ribosomal subunits in the nucleus.

Lee, Hanee et al. (2011) International Worm Meeting "Dissection of the LATS kinase pathway in C. elegans."

Kang, Junsu, Lee, Junho, Lee, Hanee

[International Worm Meeting, 2011]

The Hippo(Hpo) pathway is a conserved signaling pathway regulating organ size homeostasis via controlling cell proliferation and apoptosis. We previously reported that wts-1, the C. elegans LATS homolog which is a core gene in the Hpo pathway, showed an early larval arrest phenotype due to the defective functions of the intestine(1). In this study, we tried to find the downstream genes of wts-1 by using EMS random mutagenesis. As a result, we obtained 18 suppressor lines that suppress the larval arrest phenotype of wts-1. We identified yap-1 and egl-44, the C.elegans homolog of YAP/Yki and TEAD respectively, as suppressors of wts-1 by SNP mapping. Mapping of the remaining mutations is in progress. Because it is known that LATS kinase acts through YAP/yki, a transcription co-activator, which interacts with the TEAD transcription factor, our results suggest that the core components of the Hpo pathway and their genetic interaction are conserved in C.elegans. Therefore, C. elegans is a good model organism to study the Hpo pathway. To delineate the Hpo pathway in C. elegans, we are looking for upstream and downstream genes using RNAi screening and microarray, respectively. Our work will contribute to further understanding of the Hpo pathway. (1)Kang J, et al. (2009), Lats kinase is involved in the intestinal apical membrane integrity in the nematode Caenorhabditis elegans. Development 136(16), 2705-15.

Vida Praitis et al. (2004) Mid-west Worm Meeting "The unc-59(ru5) mutation affects morphogenesis of the pharynx"

Lensa Yohannes, Juliette Mushi, Zelealem Yilma, Michael Miller, Sarah Kniss, Vida Praitis

[Mid-west Worm Meeting, 2004]

In C. elegans , pharyngeal morphogenesis is divided into 3 discreet steps: (1) reorientation of the anterior pharyngeal cells, (2) formation of epithelial contacts between the pharyngeal cells, buccal cavity cells, and anterior hypodermis, and (3) contraction, which brings the pharynx, buccal cavity, and mouth together (Portereiko & Mango, 2001). We have identified a temperature-sensitive embryonic lethal mutation which causes defects in pharyngeal morphogenesis, resulting in embryos with the pun (pharynx unattached) phenotype. Genetic experiments show the ru5 mutation is weakly semi-dominant but maternal or zygotic product is sufficient to rescue the temperature-sensitive lethal phenotype. ru5 embryos maintained at the restrictive temperature undergo normal development until the bean stage when pharyngeal morphogenesis fails to occur properly. ru5 embryos exhibit defects that include mis-oriented anterior pharynxes, excess basement membrane anterior to the pharynx, as assayed by anti-NID-1 antisera (Kang & Kramer, 2000), and, even when the orientation of the pharynx appears normal, failure to create a stable epithelium. We believe the defects we observe are due to a failure in morphogenesis rather than altered cell fate or number because the unattached pharynx is otherwise normal as development proceeds. Temperature shift experiments show the gene product is required for viability from ~4 to 6 hours after the two cell stage, prior to pharyngeal morphogenesis. Larva and adults, grown at the permissive temperature or at the restrictive temperature after the temperature sensitive period, have phenotypes that include uncoordination and vulva morphology defects. Using conventional and snp-snp mapping strategies, we mapped the ru5 mutation to a small interval on LG I. Recent cosmid rescue and sequencing experiments have revealed the ru5 mutation is a new allele of the unc-59 septin gene. Previously identified mutations in unc-59 cause defects in distal tip cell and axon migration and post-embryonic cytokinesis (Nguyen, Sawa, Okano & White, 2000; Finger, Kopish & White, 2003). There is also a low penetrance embryonic lethality caused by failure of the pharynx to attach to the mouth (Finger, personal communication). We are currently examining the expression of UNC-59 protein in ru5 mutants grown at the restrictive temperature to characterize the nature of the unc-59(ru5) allele. We believe the defects we observe in unc-59(ru5) mutants support the model that septins may be generally required for cell migration and tissue morphogenesis.

Hill, Robin L. (2011) International Worm Meeting "Characterizing wild nematode isolates in an undergraduate research lab."

Hill, Robin L.

[International Worm Meeting, 2011]

Small Public Liberal Arts Colleges pose multiple challenges, particularly in teaching laboratory courses. Resources are limited and the majority of students have no lab experience. In addition, faculty struggle to maintain a full-time active research program due to heavy teaching loads. A significant percentage of students expressing an interest in the sciences also express a desire for graduate or professional studies. As many of these programs desire "hands-on" scientific experience, providing research opportunities could be vital to their future success. To address these issues, a full semester research-based project was developed around isolating and identifying wild nematode isolates. An overview of nematode phylogeny and population genetics studies is coupled with students working in small groups to develop a research project, isolate nematodes and characterize the isolates using morphological and molecular analysis. Results are presented in a format similar to graduate lab meetings. The molecular techniques utilized during the pilot project included species-specific PCR (glp-1) (1) as well as sequencing of the 18S ribosomal RNA gene (2). I adapted universal rice primers (URP) as a fingerprint assay (3). Morphological analysis included buccal (lips, buccal tube, pharynx) and tail (shape, papillae and spicule structure) morphology as well as mating system (gonochoristic vs. hermaphroditic). Strains from the CGC have been used for comparison (both morphological and molecular) to wild isolates. Mating crosses with C.elegans and C.briggsae males have been performed with hermaphroditic species. Males from gonochoristic strains can be employed for gonochoristic isolates. Hermaphroditic isolates were obtained from leaf litter in wooded settings, open grasslands and under a rotting pumpkin. Species-specific PCR as well as mating crosses reject identification as C.elegans or C.briggsae. Sequencing of the 18S RNA gene is ongoing. Gonorchorisitc isolates were obtained from both household and grass cutting compost. Morphological analysis based on tail morphology suggests the genus Rhabditis, subgenus Rhabditella and Cephalopoides. Student feedback was uniformly positive. Isolates were easily obtained and the self-directed nature of the projects deepened their understanding of lab methods and research design. The range of analysis methods allows the course to be adapted to resource availability, therefore providing a method of research experience while enhancing knowledge of the model species. (1) Barriere, A., and Felix M.A. (2005). Curr. Biol. 15,1176-184. (2) Floyd, R. et al. (2002). Mol Ecol. 11, 839-850. (3) Kang, H. W. et al. (2002). Mol. Cells. 2, 281-287.

Gallegos, Maria E. (2009) International Worm Meeting "A discovery-based molecular techniques course aimed to develop reagents for rab gene function studies."

Gallegos, Maria E.

[International Worm Meeting, 2009]

I have developed a discovery-based lab course at California State University, East Bay (CSUEB) entitled Advanced Molecular Techniques. This lab course has three aims: 1) to promote the National Science Education Standards mandate to replace "cookbook" lab courses with inquiry-based learning; 2) to teach a variety of molecular biology techniques applicable in both academia and industry settings and 3) to develop molecular tools useful to the C. elegans community for gene function studies. During the quarter, students learn a variety of standard molecular techniques in addition to QuikChange Site-Directed Mutagenesis (Stratagene) and Gateway Recombinational Cloning (Invitrogen). To prepare for and execute each step of the project, students must also master software tools such as NCBI BLAST (NLM), CLUSTAL W (EMBL-EBI), Primer Generator (Lawler and Turchin), A Plasmid Editor (Wayne Davis) and Image J (NIH), all of which are available free online. Students use these software tools and molecular techniques to modify Orfeome entry clones and create expression vectors containing dominant negative and constitutive active forms of the rab monomeric GTPases (entry clones kindly provided by Dr. Kang Shen). To date we have focused on the rab family of GTPases for several reasons: The rab family is manageable in size with 22 members. The average coding sequence is short, about the length of a traditional sequencing run. Dominant negative and constitutive active forms are well characterized. And finally, rab GTPases play important and diverse roles in cell and developmental biology. Each pair of students is assigned a single gene. One will create the dominant-negative while the other will create the constitutive active form. Thus each student owns a unique project with unique solutions while still having access to backup reagents (when necessary) and advice from peers. To provide each student a unique project, I needed to reduce costs. The QuikChange Site-Directed Mutagenesis kit by Stratagene is the most expensive component of this course thus each year we have also experimented with its protocol. Importantly, our results indicate that you do not need to use PAGE purified mutagenic primer pairs (as recommended), there is no reduction in mutagenesis efficiency if half the reaction volume is used and you can still get a sufficient number of transformants with half the volume of competent cells. Our progress will be reported. Any tools that have been created are freely available to the worm community.

Lee, Inhwan et al. (2011) International Worm Meeting "AMP-dependent protein kinase regulates nutrition dependent diapause in C. elegans."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2011]

To survive starvation, animals reduce their energy use by turning off anabolic pathways and burn stored fuel by turning on catabolic pathways. One of the molecular switches doing that is AMP-dependent protein kinase (AMPK). Using L1 diapause, we study how AMPK regulates the long term starvation. We found that starvation during L1 activates AMPK and induces two phenotypes in aak-2 mutants; (1) 3 day starvation as L1 induces sterility when the starvation-experienced worms become adults. (2) 7 day starvation as L1s kills them. To get fully activated, the threonine residue at 172 in AMPK needs to be phosphorylated. Because L1 starvation increases phosphorylation in AMPK, we tested the mutants of three known upstream kinases of AMPK (PAR-4, MOM-4 and CKK-1) for both the sterility and the lethality phenotypes. None fully phenocopy the phenotypes induced by L1 starvation in aak-2 mutants; only par-4 mutants partially resemble them, suggesting redundancy among these upstream kinases. Autophagy is self-degradation process for cells to survive starvation and we previously showed insufficient autophagy shortens L1 diapause span due to insufficient energy [1]. Because AMPK directly induces autophagy by phosphorylating ULK-1 [2], we hypothesized that aak-2 die earlier than wild type because the mutants fail to induce autophagy. When we measured the amount of cleaved LGG-1, a homolog of LC3 and an indicator of active autophagy, it is reduced in aak-2, suggesting that the reduction of autophagy contributes to the death of aak-2. It has been suggested that metformin, the most widely used drug for type II diabetes acts through AMPK. But its direct target(s) are unknown. To confirm the function of AMPK during L1 diapause by pharmacological approach and to test if L1 diapause can be used to find targets of metformin, we treated worms with metformin during L1 diapause. Based on the its action, we hypothesize that (1) metformin will over-activate AMPK during L1 diapause where AMPK is already activated. Therefore it will shorten L1 diapause span. (2) If metformin acts through AMPK, treatment with metformin would not shorten L1 diapause span any further in aak-2 mutants. We found both true; treatment with metformin shortens L1 diapause and it doesn't reduce the short L1 diapause span of aak-2 further. Our study shows that L1 diapause is starvation response regulated by AMPK; AMPK functions for worms to survive the long-term starvation undamaged. 1.Kang, C., Y.J. You, and L. Avery. Genes Dev, 2007. 21(17): p. 2161-71. 2.Egan, D.F., et al. Science, 2011. 331(6016): p. 456-61.