Tan, Ee Phie et al. (2019) International Worm Meeting "Probing Mechanisms of Selective Autophagy Relevant to Aging."

Hansen, Malene, Choy, Elizabeth, Tan, Ee Phie

[International Worm Meeting, 2019]

Autophagy is an evolutionarily conserved cellular recycling process, which is critical for development, disease, and aging. Therefore, determining the molecular action of autophagy is crucial to better understand organismal health. Notably, autophagy is increasingly appreciated as a selective process by which specific types of cytoplasmic material or cargo, such as mitochondria or lipids are sequestered into double-membrane vesicles called autophagosomes, which fuse with lysosomes to facilitate degradation of the contents. Selective cargo sequestration to the autophagosome is achieved by autophagy receptors, which simultaneously bind specific cargo and Atg8/LC3/GABARAP, a protein localized to the autophagosomal membrane. While accumulating evidence indicates that dysregulation in selective types of autophagy can contribute to the development of age-related diseases, the relevant selective autophagy receptors and their role in aging remain understudied. To identify selective autophagy receptors relevant to aging, we performed a candidate RNA interference (RNAi) screen using genes previously reported to be potential autophagy receptors in other model systems. We used C. elegans expressing GFP-tagged Atg8/LGG-1 and looked for aberrant Atg8/LGG-1 expression upon gene knockdown. In this primary screen, we identified many candidate genes to increase LGG-1 levels. Currently, we are performing secondary RNAi screens of candidates from the primary screen, utilizing cargo-specific reporters to look for genes that increase cargo levels upon knockdown. In a parallel approach, we are also performing unbiased proteomic screenings to identify proteins that interacts with Atg8/LGG-1 and which could be potential autophagy receptors. Proteins of interests from these screening approaches will be analyzed further by immunoprecipitation and immunohistochemistry assays to determine if they are potential autophagy receptors, and if they are critical for the healthspan and longevity of the animal. This study will improve our molecular understanding of the regulation of the physiological relevance of selective autophagy in aging. Such insights may provide new entry points for designing targeted therapeutic interventions against age-related diseases.

Tan, Ee Phie et al. (2021) International Worm Meeting "An autophagy activator extends healthspan and lifespan in C. elegans"

Lyang, Nora, Kelly, Jeffery, Xu, Jin, Hansen, Malene, Tan, Ee Phie, Nieto-Torres, Jose, Botham, Rachel, Yoon, Leonard, Zaretski, Svaitlana, Johnson, Kristen

[International Worm Meeting, 2021]

Autophagy is an evolutionarily conserved cellular recycling process with tight links to longevity and healthspan. In particular, autophagy function declines during aging, and is dysregulated in many age-related disorders such as in neurodegenerative diseases. Therefore, identifying interventions that can boost autophagy to prevent such chronic illnesses progression is crucial to improving organismal health. Of note, autophagy is increasingly appreciated as a selective process by which specific types of cytosolic cargo, such as organelles, lipids and protein aggregates, are sequestered into double-membrane structures called autophagosomes that subsequently fuse with hydrolase-containing lysosomes to enable cargo degradation. Interestingly, accumulating evidence suggests that disruptions in selective autophagy can contribute to the development of age-related diseases. For example, chronic inhibition of lipophagy (selective lipid turnover) leads to increased accumulation of lipids, leading to obesity and diabetes. However, treatments that may target and improve selective autophagy to help relieve such illnesses remain underdeveloped. To identify novel chemical compounds that may act as selective autophagy activators, we performed a high-throughput imaging screen in human adenocarcinoma cells to uncover small molecules that activate autophagy and increase lipid clearance. Given the previous links between autophagy and aging, we tested several autophagy activator hit compounds from the screen for autophagy- and lifespan assays in C. elegans. While we found that these compounds all increased autophagosome numbers, only animals fed with small molecule A20 exhibited life- and healthspan extension, along with reduced lipid levels, as observed in human cells. Importantly, this A20-mediated lipid reduction and health benefits were not observed in autophagy mutants. Furthermore, we found that A20 could reduce PolyQ aggregate cargo load in multiple tissues, and we are currently investigating if A20 is affecting additional cytosolic cargos. Notably, inhibition of the nutrient sensor mTORC1 activates autophagy. However, A20 seemed to function independently of mTORC1, and we are currently performing studies to determine how A20 could mediate autophagy. In conclusion, we have identified a new compound A20, which may potentially be applied in future strategies to improve organismal health and alleviate age-related diseases by boosting autophagy.

Ramamurthy Mani et al. (2005) International Worm Meeting "Predictive models of molecular machines involved in C. elegans early embryogenesis"

Christophe Echeverri, Anthony Hyman, Ramamurthy Mani, Kristin C Gunsalus, Ning Li, Ling-Shiang Chuang, Marc Vidal, Fabio Piano, Brite Sonnichsen, Frederic P Roth, Debra S Goldberg, Aaron J Schetter, Nicolas Bertin, Jing-dong J Han, Jerry Huang, Hui Ge, Gabriel Berriz

[International Worm Meeting, 2005]

While numerous fundamental aspects of development have been uncovered through the discovery of individual genes and proteins, systems-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans constitute an ideal testbed for a systems-level approach. Early embryogenesis (EE), including processes like cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a systems-level understanding is to provide first-draft models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationships: protein interaction, expression profiling similarity, and phenotypic profiling similarity, as estimated from detailed early embryonic RNAi phenotypes systematically recorded for hundreds of EE genes. The topology of the integrated network suggests that C. elegans EE is achieved through coordination of a limited set of molecular machines. The overall predictive value of such molecular machine models was assessed by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Kenji Kimura et al. (2008) "A tug-of-war model for the centrosomal centration in C. elegans early embryo."

Kenji Kimura, Akatsuki Kimura

[2008]

"The centrosome is the microtubule (MT) organizing center and critical for MT dependent cellular functions in animal cells. The position of the centrosome is actively maintained at the cell center. Male pronuclear migration (PNM), the migration of male pronucleus-centrosome complex from periphery to center of the cell after fertilization, is a typical example of the centrosomal centration. As a primary centrosomal centration mechanism, it has reported that the centration requires the MT pulling forces by the minus-end directed motor protein, cytoplasmic dynein. However, it remains unclear where the MT is pulled and how the pulling force is generated by the dynein during PNM. Here, we propose a ""tug-of-war model"" that the dynein dependent minus-end directed organelle movement along the MT generates the MT pulling force for the centration. Three lines of observations during PNM in C. elegans support this model. First, we observed the subcellular localization of the dynein using GFP-fused dynein subunits to examine where the dynein functions during PNM. These reporter proteins localized to cytoplasm and the MT elongated from the centrosome during PNM. We found that the localization on the MT is dependent on dhc-1 which encodes dynein heavy chain subunit and is essential for motor activity. The result suggests that the dynein on the MT are generating the pulling force during PNM. Second, we quantified transport of some organelles along the MT during PNM to analyze the correlation between the dynein dependent organelle movement and PNM. We found that early endosome (EE) moved straightly toward the centrosome in a dhc-1 dependent manner. In addition, the direction of total vector of EE movements is opposite to the direction of PNM. The result suggests that an inverse correlation exists between EE movement and PNM. Third, we addressed whether PNM is inhibited by reduction of EE movement to verify our model. We found that the rate of PNM was delayed by knockdown of some transport regulators. These results suggest that dynein dependent movements of organelles toward the centrosome generate a counteracting force as the MT pulling force, and contribute to the centrosomal centration during PNM in C. elegans."

Huang, Huiyan et al. (2021) International Worm Meeting "C. elegans modeling and human studies of a rare RAB5B patient variant reveal a novel role of RAB5B in regulated secretion of pulmonary surfactant"

Huang, Huiyan, Lindsey, Anika, Dawson, Zachary, Spielberg, David, Chen, Jian, Schedl, Tim, Mohammad, Ariz, Burrage, Lindsay, Kim, Hyori, Baldridge, Dustin, Pan, Jiehong, Rosenfeld, Jill, Scott, Daryl, Pak, Stephen, Hanchard, Neil, Murdock, David, Morrison, Stephanie, Dai, Hongzheng, Brody, Steven, Zhu, Alex, Tycksen, Eric, Silverman, Gary, Ramu, Avinash

[International Worm Meeting, 2021]

Many patients with severe, chronic diseases remain without a diagnosis despite extensive medical evaluation, including some cases with clinical exome sequencing. The goal of the NIH-funded Undiagnosed Diseases Network (UDN) is to provide a diagnosis for these challenging cases and to identify biological characteristics of newly discovered disease genes. The UDN uses a collaborative multidisciplinary approach that combines comprehensive medical workup, exome/genome sequencing, bioinformatic analysis, with functional studies in model organisms including zebrafish, Drosophila, and C. elegans. Through the UDN, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant, p.Asp136His, in the Ras/Rab GTPases family nucleotide binding domain in RAB5B was identified. Functional studies were performed in the C. elegans Model Organism Screening Center at Washington University in St. Louis. We used CRISPR/Cas9 to knock the proband variant into the conserved position (Asp135) of the ortholog, rab-5. Analyses of organismal phenotypes such as locomotion and size demonstrated that rab-5[Asp135His] is damaging. We also show data indicating that rab-5[Asp135His] heterozygotes were defective in endocytosis and early endosome (EE) fusion. Dosage analysis by adding extra copies of wild type rab-5 transgene revealed that rab-5[D135H] has a strong dominant negative effect, requiring three wild type copies to suppress the variant's poisonous effect. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in type II pneumocytes, and mature SP-B and SP-C were significantly reduced, while ProSP-B and ProSP-C were normal. Furthermore, staining of normal lung showed co-localization of RAB5B and EEA1 with ProSP-B and ProSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, inducing interstitial lung disease, and that RAB5B and EEs normally function in the regulated surfactant secretion pathway. Together, the data suggest a non-canonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for surfactant dysfunction disorder.

Michael Brauchle et al. (2007) International Worm Meeting "Evolution of early embryonic developmental mechanisms in Rhabditidae."

Fabio Piano, Michael Brauchle

[International Worm Meeting, 2007]

Natural diversity ultimately arises from the evolution of developmental programs. The molecular mechanisms underlying the evolution of developmental programs are still largely mysterious. A critical question is thus to discover mechanisms that enable evolution of developmental programs in light of the highly constrained mechanistic view that arises from developmental genetics, where altering important developmental genes is known to lead to lethality. We are using nematode early embryogenesis (EE) as a model to study these questions. When analyzed by time-lapse microscopy, nematode embryos show a remarkable array of different characteristics during EE. To analyze this diversity in more detail, we have developed a series of 40 discrete phenotypic characters describing the first two rounds of cell division among 35 Rhabditidae species. When all the characters are mapped onto a molecular phylogeny we find that they have evolved multiple times, showing a high rate of convergent evolution. This observation supports the idea that early development is plastic. The diversity in EE is surprising given the reproducible way in which each single species, like C. elegans, develop. One possible reason for this evolutionary plasticity is that early development is governed by highly modular molecular mechanisms that can direct specific events independently from one another. In support of this idea, we find almost no evidence for correlation (co-evolution) among characters across the 35 species. To obtain molecular clues that could underlie the diversity we observed, we compared the evolutionary characters with the phenotypes observed from RNAi data in C. elegans. Surprisingly, for almost every character we could find a matching RNAi phenotype that phenocopies the character of another species. One hypothesis that arose from these RNAi comparisons was that a rotation of the spindle in the AB cell seen in Protorhabditis and Diploscapter species was due to a change in the polarity network. The polarity network is defined by the regulatory interactions among the PAR proteins in C. elegans. These comparisons led us to postulate that the PAR-3/PAR-6/PKC-3 complex may be missing or inactive in Diploscapter or Protorhabditis one-cell embryos. We tested this hypothesis by localizing PAR-1 in these species and found a novel localization pattern that support the idea that the PAR network is evolving across these species.

P. Giselle Cipriani et al. (2007) International Worm Meeting "Mapping genetic interactions in C. elegans early embryogenesis."

Fabio Piano, P. Giselle Cipriani, Kristin Gunsalus

[International Worm Meeting, 2007]

Current genome annotation estimates that the C. elegans genome contains approximately 20,000 protein-coding genes of which ~15% are essential for viability. However, less than 1,000 genes appear to be required for discrete events during early embryogenesis (EE), despite evidence that as much as half the transcriptome is present at these stages. A prevailing theory to account for these observations is that essential functions might be masked by genetic redundancy involving gene or pathway compensation. Screens for epistatic interactions have been very valuable in finding modifiers of essential genes (which may not be essential on their own), and unraveling the regulatory pathways in which they are involved. Towards a first-draft view of the genetic interactions map in C. elegans early embryogenesis we are reducing the function of two genes at the same time. Our strategy is to alter the function of an essential gene (query gene) using temperature sensitive (ts) alleles, and to use RNAi for a set of 500 genes (target genes) to reduce the function of a second gene. Using carefully selected temperatures for each ts allele, we are identifying enhancers and suppressors for each query gene. To obtain an initial global view of the genetic interaction map, we selected the set of query genes to cover all available ts alleles at the Caenorhabditis Genetics Center that give rise to EE defects. Our progress from these screens will be presented.

Catherine A Mastroieni et al. (2004) West Coast Worm Meeting "ANALYSIS OF MAP KINASE PHOSPHORYLATION SITES IN THE C. ELEGANS TRANSCRIPTION FACTOR LIN-31"

Corey A Morris, Catherine A Mastroieni, Leilani M Miller

[West Coast Worm Meeting, 2004]

Complex signaling pathways are required for the proper specification of cell fates. In the C. elegans hermaphrodite, six cells of initially equivalent developmental potential respond differentially to a Ras/MAP kinase signaling pathway, correctly choosing their respective fates to form the adult vulva. Activation of the Ras/MAP kinase pathway culminates in the phosphorylation of one or more transcription factors by MAP kinase, resulting in the induction of fate-specific genes. One of the transcription factors phosphorylated by MAP kinase is LIN-31, a winged-helix transcription factor (Miller et al., Genes Dev. 7:933, 1993) that contains four consensus MAP kinase phosphorylation sites. Co-immunoprecipitation experiments by Tan et al. (Cell 93:569, 1998) showed that LIN-31 forms a heterodimer with the LIN-1 ETS transcription factor and that this heterodimer is disrupted upon phosphorylation of LIN-31 by MAP kinase. These results support the following model in which LIN-31 has two functions: 1) in its unphosphorylated, dimerized form LIN-31 promotes non-vulval cell fates, and 2) in its phosphorylated, undimerized form LIN-31 promotes vulval cell fates. Consistent with the model that phosphorylation of the MAP kinase consensus sites is necessary for LIN-31 function in promoting vulval cell fates, previous studies (Tan et al., 1998) have shown that deletion of all four phosphorylatable sites in LIN-31 results in defective vulval formation. Since the core sequence for MAP kinase consensus sites (S/T-P) consists of only two amino acids, many "consensus" sites identified by sequence alone may not actually be phosphorylated by MAP kinase. Although the first MAP kinase phosphorylation site in the LIN-31 protein is a "full" site (P-X-S/T-P) and Tan et al. (1998) showed that removal of this site resulted in a 50% decrease in overall phosphorylation of the LIN-31 protein, the other three sites contain only the "core" sequence. We are using site-directed mutagenesis, protein expression and purification, and phosphorylation experiments to investigate the phosphorylation pattern of the putative MAP kinase phosphorylation sites in the LIN-31 protein.

Ewbank JJ et al. (2000) European Worm Meeting "Using C. elegans to identify Serratia marcescens virulence factors"

Ewbank JJ, Kurz CL, Finlay BB, Celli J

[European Worm Meeting, 2000]

Tan and Ausubel, working with the enterobacterium Pseudomonasaeruginosa, have established C. elegans as a model for the study of pathogenesis and host defences. Clearly, the use of the worm as a model for studying pathogenicity will be limited to those pathogens that are able to infect the worm. Luckily, in this respect, its susceptibility to P. aeruginosaappears not to be an isolated case. A second opportunistic human pathogen, Serratia marcescens, is also capable of infecting C. elegans. Like P. aeruginosa, S. marcescens is able to infect a broad range of plant and animal hosts and has been used as a model pathogen in studies of Drosophila innate immunity. Using a strain of S. marcescensthat expresses GFP, we have been able to follow the infection process. The bacteria are able to survive within the usually hostile environment of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens. We are currently screening these mutant bacterial clones individually for those showing reduced virulence.Of the first 2000 mutants screened, 18 showing markedly reduced virulence have been retained for further study. The molecular characterization of these mutants may reveal novel virulence factors that represent potential drug targets. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. S: 29-34.

C Leopold Kurz et al. (2001) International C. elegans Meeting "Identifying universal Serratia marcescens virulence factors using C. elegans."

Jonathan Ewbank, Jean Celli, Dominique Ferrandon, B Brett Finlay, Emmanuel Andres, Sophie Chauvet, C Leopold Kurz, Mitchell Uh

[International C. elegans Meeting, 2001]

Tan and Ausubel, working with the enterobacterium Pseudomonas aeruginosa , have established C. elegans a s a model for the study of pathogenesis and host defences . A second opportunistic human pathogen, Serratia marcescens , is also capable of infect ing C. elegan s . Like P. aeruginosa , S. marcescens is able to infect a broad range of plant and animal hosts. Using a strain of S. marcescens that expresses GFP, we have been able to follow the infection process. T he bacteria are able to survive within the usually hostile environmen t of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens . We are currently screening these mutant bacterial clones individually for those showing reduced virulence . Of the first 2000 mutants screened, 9 showing markedly reduced virulence have been retained for further study. T he molecular characterization of these mutants has revealed novel virulence factors. In order to determine whether these virulence factors are specific to the infection of the nematode, we have also tested them in a Drosophila infection model and found that 4 of them are attenuated for their virulence. Tests in a mammalian model will reveal whether we have identified virulence factors that are important irrespective of the host. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans : a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 3: 29-34.