189 Papers found (0.01 seconds)

Seroussi U et al. (2023) Elife "A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions."

Claycomb JM, Zhao W, Charlesworth AG, Lugowski A, Seroussi U, Lao RX, Sundby AE, Wadi L, Reinke AW, Willis AR

[

Elife,

2023]

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode C. elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work we systematically analyzed every C. elegans AGO, using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

Nehammer C et al. (2019) Elife "Interferon--induced miR-1 alleviates toxic protein accumulation by controlling autophagy."

Nehammer C, Rubinsztein DC, Gopal S, Ng L, Handley A, Ejlerskov P, Pocock R, Lee H, Moreira P, Issazadeh-Navikas S

[

Elife,

2019]

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous Tre-2/Bub2/CDC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in Caenorhabditis elegans and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-b (IFN-b) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders.

He S et al. (2019) Genetics "NATF (Native And Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans."

Miller DM, He S, Cuentas-Condori A

[

Genetics,

2019]

GFP labeling by genome editing can reveal the authentic location of a native protein but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy which combines CRISPR/Cas-9 and split-GFP to yield bright, cell-specific protein labeling. We use CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 B-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein, OIG-1, and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the C. elegans nervous system.

Charlesworth AG et al. (2019) Cell "Next-Gen Learning: The C.elegans Approach."

Claycomb JM, Seroussi U, Charlesworth AG

[

Cell,

2019]

In this issue, Moore etal. and Posner etal., provide evidence for how the activity of the nervous system in C.elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.

Renaud MS et al. (2023) Methods Mol Biol "Analysis of C. elegans Germline Small RNA Pathways."

Renaud MS, Claycomb JM, Seroussi U

[

Methods Mol Biol,

2023]

Sequence-specific gene regulation by small RNA (sRNA) pathways is essential for the development and function of organisms in all domains of life. These regulatory complexes, containing an Argonaute protein (AGO) guided by a bound sRNA, have the potential to regulate thousands of individual target transcripts at both the co- and post-transcriptional level. Determining the repertoire of transcripts that an AGO is capable of regulating in a particular context is essential to understanding the function of these regulatory modules. Immunoprecipitation (IP) of AGOs and subsequent RNA sequencing of their bound sRNAs allows for the inference of their target transcripts by mapping the sequences of the co-precipitated sRNAs back to their complementary target transcripts. This approach can be complemented by sequencing sRNAs from ago mutants as sRNA transcripts are degraded in the absence of their AGO binding partner. Here, we describe a framework for analyzing AGO/sRNA pathways in the germline, from using CRISPR-Cas9 to tag or mutate AGOs, through protocols for the extraction, sequencing, and analysis of sRNAs from AGO IPs and ago mutants.

Gorelick S et al. (2019) Elife "PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy."

Gervinskas G, Whisstock JC, Handley A, Johnson TK, Buckley G, Caggiano MP, de Marco A, Gorelick S, Pocock R

[

Elife,

2019]

Cryo-Electron Tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Nithianantham S et al. (2018) J Biol Chem "Structural basis for disassembly of katanin heterododecamers."

Nithianantham S, McNally FJ, Al-Bassam J

[

J Biol Chem,

2018]

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

Dutilleul M et al. (2013) Ecotoxicology "Rapid phenotypic changes in Caenorhabditis elegans under uranium exposure."

Lecomte C, Lemaire L, Bonzom JM, Dutilleul M, Reale D, Galas S

[

Ecotoxicology,

2013]

Pollutants can induce selection pressures on populations, and the effects may be concentration-dependant. The main ways to respond to the stress are acclimation (i.e. plastic changes) and adaptation (i.e. genetic changes). Acclimation provides a short-term response to environmental changes and adaptation can have longer-term implications on the future of the population. One way of studying these responses is to conduct studies on the phenotypic changes occurring across generations in populations experimentally subjected to a selective factor (i.e. multigenerational test). To our knowledge, such studies have not been performed with uranium (U). Here, the phenotypic changes were explored across three generations in experimental Caenorhabditis elegans populations exposed to different U-concentrations. Significant negative effects of U were detected on survival, generation time, brood size, body length and body bend. At lower U-concentrations, the negative effects were reduced in the second or the third generation, indicating an improvement by acclimation. In contrast, at higher U-concentrations, the negative effects on brood size were amplified across generations. Consequently, under high U-concentrations acclimation may not be sufficient, and adaptation of individuals would be required, to permit the population to avoid extinction. The results highlight the need to consider changes across generations to enhance environmental risk assessment related to U pollution.

Mok C et al. (2020) G3 (Bethesda) "PhenoMIP: High-Throughput Phenotyping of Diverse Caenorhabditis elegans Populations via Molecular Inversion Probes."

Moerman DG, Waterston RH, Belmarez G, Edgley ML, Mok C

[

G3 (Bethesda),

2020]

Whether generated within a lab setting or isolated from the wild, variant alleles continue to be an important resource for decoding gene function in model organisms such as Caenorhabditis elegans With advances in massively parallel sequencing, multiple whole-genome sequenced (WGS) strain collections are now available to the research community. The Million Mutation Project (MMP) for instance, analysed 2007 N2-derived, mutagenized strains. Individually, each strain averages ~400 single nucleotide variants amounting to ~80 protein-coding variants. The effects of these variants, however, remain largely uncharacterized and querying the breadth of these strains for phenotypic changes requires a method amenable to rapid and sensitive high-throughput analysis. Here we present a pooled competitive fitness approach to quantitatively phenotype subpopulations of sequenced collections via molecular inversion probes (PhenoMIP). We phenotyped the relative fitness of 217 mutant strains on multiple food sources and classified these into five categories. We also demonstrate on a subset of these strains, that their fitness defects can be genetically mapped. Overall, our results suggest that approximately 80% of MMP mutant strains may have a decreased fitness relative to the lab reference, N2. The costs of generating this form of analysis through WGS methods would be prohibitive while PhenoMIP analysis in this manner is accomplished at less than one-tenth of projected WGS costs. We propose methods for applying PhenoMIP to a broad range of population selection experiments in a cost-efficient manner that would be useful to the community at large.

Wu D et al. (2008) J Gerontol A Biol Sci Med Sci "The U-shaped response of initial mortality in Caenorhabditis elegans to mild heat shock: does it explain recent trends in human mortality?"

Cypser JR, Johnson TE, Yashin AI, Wu D

[

J Gerontol A Biol Sci Med Sci,

2008]

U-shaped dose-response relationships (hormesis) have been documented in numerous biological, toxicological, and pharmacological investigations. For example, in response to a mild 35 degrees C heat shock, the longevity of Caenorhabditis elegans exhibits an inverted U-shaped dose-response. By applying the demographic concept of heterogeneity, we find that this U-shaped curve for longevity response is driven by a U-shaped dose-response of initial mortality. When worms are subjected to mild heat shock, the initial mortality decreases compared to the control. This initial mortality benefit increases with moderate increases in the length of heat shock, peaking at a point that coincides with the induction of damage to the worms. The dose of heat shock that coincided with this benefit in initial mortality did not affect the rate of increase in mortality.

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