(2024) Development "The people behind the papers - Nadia Manzi and Daniel Dickinson."

[Development, 2024]

The mitotic kinase Aurora A has been shown to regulate the anterior-posterior polarity in developing Caenorhabditis elegans embryos. In a new study, Daniel Dickinson and colleagues find that Aurora A has temporally distinct roles in coordinating the localization of Partitioning defective (PAR) proteins to establish cell polarity during development. To find out more about the story behind the paper, we caught up with first author Nadia Manzi and corresponding author Daniel Dickinson, Assistant Professor at the University of Texas at Austin.

Chen X et al. (2015) Mol Neurodegener "Erratum to: Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression."

McCue HV, Chen X, Morgan A, Kashyap SS, Barclay JW, Kraemer BC, Burgoyne RD, Wong SQ

[Mol Neurodegener, 2015]

The original version of this article [1] unfortunately contained a mistake. The author list contained a spelling error for the author Hannah V. McCue. The original article has been corrected for this error. The corrected author list is given below:Xi Chen, Hannah V. McCue, Shi Quan Wong, Sudhanva S. Kashyap, Brian C. Kraemer, Jeff W. Barclay, Robert D. Burgoyne and Alan Morgan

King, Skylar et al. (2015) International Worm Meeting "Ceramide Biosynthesis Impacts Stress Responses in C.elegans- A Model for Mitochondrial Dysfunction."

Quan, Daniel, Azad, Rajeev, King, Skylar, Padilla, Pamela

[International Worm Meeting, 2015]

Mitochondrial function is essential for regulating many processes such as metabolism and responses to stress. An excess of ROS can damage macromolecules, alter signaling-pathways, and promote mitochondrial dysfunction. Inducers of ROS include external toxins, environmental stressors, metabolic stress and genetic mutations. The biosynthesis of ceramide lipids is known to impact ROS levels, metabolism, apoptosis, and is implicated in disease processes such as diabetes. However, the mechanistic role ceramide metabolism has on mitochondrial function is not completely understood. Our objective is to use C. elegans to determine how disruption of ceramide biosynthesis impacts stress responses and mitochondrial function in a whole animal system. Others have shown that a mutation in hyl-2, which codes for a ceramide biosynthetic enzyme, induces anoxia sensitivity and alters the pool of ceramide species. To investigate how the modulation of ceramide impacts stress responses we conducted phenotype analysis on the hyl-2(tm2031) mutant, conduced a forward genetic suppressor screen in hyl-2(tm2031) animals and used RNA-sequencing analysis to identify gene expression in the hyl-2(tm2031) mutant relative to wild-type. We found that the hyl-2(tm2031) mutant shows some resistance to the ROS generator paraquat yet is sensitive to anoxia. Further phenotype analysis of the hyl-2 mutant indicates that developmental stage and sex influences the role ceramide biosynthesis has on anoxia sensitivity. The forward genetic screening approach identified ten suppressors of the hyl-2(tm2031) anoxia sensitivity phenotype (hans). The hans mutants are also resistant to long-term anoxia. This genetic approach will likely reveal novel signaling pathways that interact with ceramide biosynthesis to respond to the stress of oxygen deprivation. Genetic mapping and whole genome sequencing is being used to identify the mutation responsible for the hans phenotype. To identify genes that are differentially expressed in the hyl-2(tm2031) mutant, relative to wild-type, we used RNA-sequencing. We will present the gene classes identified by the RNA-sequencing analysis and propose biological functions that are impacted by the abnormal regulation of ceramides. By using a genetic approach we are identifying processes that are vital for surviving severe oxygen deprivation. This work could reveal how ceramide species impact mitochondrial dysfunction, stress responses and anoxia survival. .

(2019) Development "The people behind the papers - Daniel Osorio, Elaine Chan, Joana Saramago and Ana Carvalho."

[Development, 2019]

Animal cytokinesis is driven by an actomyosin ring that assembles at the cell equator and constricts to physically separate the two daughters. Although myosin is known to be essential for cytokinesis in multiple systems, whether this requirement reflects its motor or actin crosslinking activities has recently been a matter of contention. A new paper in Development now addresses this problem using the first divisions of the <i>Caenorhabditis elegans</i> embryo as a model. We caught up with the paper's three first authors Daniel Osorio, Elaine Chan and Joana Saramago, and their supervisor Ana Carvalho, Principal Investigator at the University of Porto's i3S consortium, to find out more about the story.

Vo, An A. et al. (2021) MicroPubl Biol

Ward, Jordan D., Ragle, James Matthew, Levenson, Max T., Vo, An A.

[MicroPubl Biol, 2021]

The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Yu YB et al. (2010) PLoS One "Cinnamomum cassia bark in two herbal formulas increases life span in Caenorhabditis elegans via insulin signaling and stress response pathways."

Dosanjh L, Shim BS, Yu YB, Luo Y, Tan M, Lao L

[PLoS One, 2010]

BACKGROUND: Proving the efficacy and corresponding mode of action of herbal supplements is a difficult challenge for evidence-based herbal therapy. A major hurdle is the complexity of herbal preparations, many of which combine multiple herbs, particularly when the combination is assumed to be vitally important to the effectiveness of the herbal therapy. This issue may be addressed through the use of contemporary methodology and validated animal models. METHODS AND PRINCIPAL FINDINGS: In this study, two commonly used traditional herbal formulas, Shi Quan Da Bu Tang (SQDB) and Huo Luo Xiao Ling Dan (HLXL) were evaluated using a survival assay and oxidative stress biomarkers in a well-established C. elegans model of aging. HLXL is an eleven herb formula modified from a top-selling traditional herbal formula for the treatment of arthritic joint pain. SQDB consists of ten herbs often used for fatigue and energy, particularly in the aged. We demonstrate here that SQDB significantly extend life span in a C. elegans model of aging. Among all individual herbs tested, two herbs Cinnamomum cassia bark (Chinese pharmaceutical name: Cinnamomi Cortex, CIN) and Panax ginseng root (Chinese pharmaceutical name: Ginseng Radix, GS) significantly extended life span in C. elegans. CIN in both SQDB and HLXL formula extended life span via modulation of multiple longevity assurance genes, including genes involved in insulin signaling and stress response pathways. All the life-span-extending herbs (SQDB, CIN and GS) also attenuated levels of H2O2 and enhanced small heat shock protein expression. Furthermore, the life span-extending herbs significantly delayed human amyloid beta (Abeta)-induced toxicity in transgenic C. elegans expressing human Abeta. CONCLUSION/SIGNIFICANCE: These results validate an invertebrate model for rapid, systematic evaluation of commonly used Chinese herbal formulations and may provide insight for designing future evidence-based herbal therapy(s).

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223

Astrid Kleinert et al. (2006) European Worm Meeting "Analysis of Insulin-Like Peptides in C. elegans by Mass Spectrometry"

Astrid Kleinert, Daniel Hess, Joy Alcedo, Jan Hofsteenge

[European Worm Meeting, 2006]

Astrid Kleinert, Daniel Hess, Jan Hofsteenge and Joy Alcedo. Genetic analyses in C. elegans have implicated an endocrine signaling pathway, the insulin/IGF-1 pathway, in regulating lifespan. Mutations in the gene daf-2, which encodes an insulin/IGF-1 receptor homologue, can increase worm lifespan by more than 100% (1). In addition, the insulin and IGF-1 pathways have been shown to influence fly and mouse lifespan (2-5).. Although downstream components of the DAF-2 pathway have been studied in detail, far less is known about the putative DAF-2 ligands predicted to be encoded by 38 insulin-like genes in the worm. It remains unknown which of the predicted insulin-like peptides are present in wild type or in longevity mutants. In order to determine this, we have initiated a project to analyze the polypeptide subfractions of the C. elegans proteome by mass spectrometry. Worm lysates will be prepared from mixed stage, wild-type worms, excluding membrane-bound proteins. Acid-ethanol extraction and gel filtration will be tested as methods to obtain a subfraction enriched in small polypeptides. Peptide identification will be done by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data analyses will be carried out using a MASCOT search database, which contains a subset of the UNIPROT database and a database generated of relevant C. elegans polypeptides. Finally, using the SILAC method for quantification (6), we plan to compare differences in the levels of polypeptides, e.g., insulin-like peptides, between wild-type worms and longevity mutants. . References: (1) Kenyon et al., 1993. Nature 366: 461-4. (2) Clancy et al., 2001. Science 292: 104-6. (3) Tatar et al., 2001. Science 292: 107-110. (4) Holzenberger et al., 2003. Nature 421: 182-7. (5) Blher et al., 2003. Science 299:572-4. (6) Krijgsveld et al., 2003. Nature Biotech. 21: 927-931.

Heinze, Svenia.D. et al. (2019) International Worm Meeting "Permissive and instructive functions of LIN-39 during Pn.p cell proliferation."

Heinze, Svenia.D., Hajnal, Prof. Alex

[International Worm Meeting, 2019]

The Caenorhabditis elegans vulva is an excellent model to study how cell proliferation is regulated during animal development. During the first larval stage, six equivalent vulval precursor cells (VPCs) are born. These cells remain arrested in the G1 phase of the cell cycle until the beginning of the third larval stage, when their fates are specified and the three proximal VPCs proliferate to generate exactly 22 vulval cells. It is yet unknown how the exit of the 22 terminally differentiated vulval cells from the cell cycle is established and maintained throughout adulthood. The hox gene lin-39is necessary to maintain the VPCs proliferating by regulating the expression of the two cell-cycle regulators cye-1(cyclin E) and cdk-4 (Roiz et al. 2016). Accordingly, the VPCs cease to divide as soon as the levels of LIN-39 drop below a certain threshold level when the animals reach the fourth larval (L4) stage. We have used the CRISPR-Cas9 system to generate a conditional knock-down allele of lin-39 that allows us to induce LIN-39 protein degradation at different stages during vulval cell proliferation. We observed that secondary (2 deg ) VPCs lacking LIN-39 prematurely exit from the cell cycle as they undergo only two instead of three rounds of cell division. Furthermore, using VPC-specific lin-39 over-expression we observed that Pn.p cells expressing LIN-39 enter the cell cycle prematurely during the L2 stage, generating a hyperplastic phenotype with an excess number of Pn.p cells. These results indicate the LIN-39 is necessary and sufficient to promote VPC proliferation in larvae. Nevertheless, vulval cells overexpressing LIN-39 exit the cell cycle after the L4 stage, indicating that LIN-39 is not sufficient to keep somatic cells proliferating in adults. Exploiting the hyperplastic phenotype of the animals over-expressing LIN-39 as a genetic background we are searching for factors that are required to maintain a prolonged proliferating state in adult somatic cells. References: Roiz, Daniel, et al. "The C. elegans hox gene lin-39 controls cell cycle progression during vulval development." Developmental biology 418.1 (2016): 124-134.