The people behind the papers - Nadia Manzi and Daniel Dickinson.

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.

A diet of oxidative stress-adapted bacteria improves stress resistance and lifespan in C. elegans via p38-MAPK.

Organisms across taxa face stresses including variable temperature, redox imbalance, and xenobiotics. Successfully responding to stress and restoring homeostasis are crucial for survival. Aging is associated with a decreased stress response and alterations in the microbiome, which contribute to disease development. Animals and their microbiota share their environment; however, microbes have short generation time and can rapidly evolve and potentially affect host physiology during stress. Here, we leverage Caenorhabditis elegans and its simplified bacterial diet to demonstrate how microbial adaptation to oxidative stress affects the host's lifespan and stress response. We find that worms fed stress-evolved bacteria exhibit enhanced stress resistance and an extended lifespan. Through comprehensive genetic and metabolic analysis, we find that iron in stress-evolved bacteria enhances worm stress resistance and lifespan via activation of the mitogen-activated protein kinase pathway. In conclusion, our study provides evidence that understanding microbial stress-mediated adaptations could be used to slow aging and alleviate age-related health decline.

An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress.

Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

Bacterial peptidoglycan acts as a digestive signal mediating host adaptation to diverse food resources in C. elegans.

Food availability and usage is a major adaptive force for the successful survival of animals in nature, yet little is known about the specific signals that activate the host digestive system to allow for the consumption of varied foods. Here, by using a food digestion system in C. elegans, we discover that bacterial peptidoglycan (PGN) is a unique food signal that activates animals to digest inedible food. We identified that a glycosylated protein, Bacterial Colonization Factor-1 (BCF-1), in the gut interacts with bacterial PGN, leading to the inhibition of the mitochondrial unfolded protein response (UPRmt) by regulating the release of Neuropeptide-Like Protein (NLP-3). Interestingly, activating UPRmt was found to hinder food digestion, which depends on the innate immune p38 MAPK/PMK-1 pathway. Conversely, inhibiting PMK-1 was able to alleviate digestion defects in bcf-1 mutants. Furthermore, we demonstrate that animals with digestion defects experience reduced natural adaptation capabilities. This study reveals that PGN-BCF-1 interaction acts as "good-food signal" to promote food digestion and animal growth, which facilitates adaptation of the host animals by increasing ability to consume a wide range of foods in their natural environment.

Mitochondrial energy state controls AMPK-mediated foraging behavior in C. elegans.

Organisms surveil and respond to their environment using behaviors entrained by metabolic cues that reflect food availability. Mitochondria act as metabolic hubs and at the center of mitochondrial energy production is the protonmotive force (PMF), an electrochemical gradient generated by metabolite consumption. The PMF serves as a central integrator of mitochondrial status, but its role in governing metabolic signaling is poorly understood. We used optogenetics to dissipate the PMF in Caenorhabditis elegans tissues to test its role in food-related behaviors. Our data demonstrate that PMF reduction in the intestine is sufficient to initiate locomotor responses to acute food deprivation. This behavioral adaptation requires the cellular energy regulator AMP-activated protein kinase (AMPK) in neurons, not in the intestine, and relies on mitochondrial dynamics and axonal trafficking. Our results highlight a role for intestinal PMF as an internal metabolic cue, and we identify a bottom-up signaling axis through which changes in the PMF trigger AMPK activity in neurons to promote foraging behavior.

In Vitro and In Vivo Insights into a Broccoli Byproduct as a Healthy Ingredient for the Management of Alzheimer's Disease and Aging through Redox Biology.

Broccoli has gained popularity as a highly consumed vegetable due to its nutritional and health properties. This study aimed to evaluate the composition profile and the antioxidant capacity of a hydrophilic extract derived from broccoli byproducts, as well as its influence on redox biology, Alzheimer's disease markers, and aging in the Caenorhabditis elegans model. The presence of glucosinolate was observed and antioxidant capacity was demonstrated both in vitro and in vivo. The in vitro acetylcholinesterase inhibitory capacity was quantified, and the treatment ameliorated the amyloid-ß- and tau-induced proteotoxicity in transgenic strains via SOD-3 and SKN-1, respectively, and HSP-16.2 for both parameters. Furthermore, a preliminary study on aging indicated that the extract effectively reduced reactive oxygen species levels in aged worms and extended their lifespan. Utilizing broccoli byproducts for nutraceutical or functional foods could manage vegetable processing waste, enhancing productivity and sustainability while providing significant health benefits.

Role of GLR-1 in Age-Dependent Short-Term Memory Decline.

As the global elderly population grows, age-related cognitive decline is becoming an increasingly significant healthcare issue, often leading to various neuropsychiatric disorders. Among the many molecular players involved in memory, AMPA-type glutamate receptors are known to regulate learning and memory, but how their dynamics change with age and affect memory decline is not well understood. Here, we examined the in vivo properties of the AMPA-type glutamate receptor GLR-1 in the AVA interneuron of the Caenorhabditis elegans nervous system during physiological aging. We found that both total and membrane-bound GLR-1 receptor levels decrease with age in wild-type worms, regardless of their location along the axon. Using fluorescence recovery after photobleaching, we also demonstrated that a reduction in GLR-1 abundance correlates with decreased local, synaptic GLR-1 receptor dynamics. Importantly, we found that reduced GLR-1 levels strongly correlate with the age-related decline in short-term associative memory. Genetic manipulation of GLR-1 stability, by either deleting msi-1 or expressing a ubiquitination-defective GLR-1 (4KR) variant, prevented this age-related reduction in receptor abundance and improved the short-term memory performance in older animals, which reached performance levels similar to those of young animals. Overall, our data indicate that AMPA-type glutamate receptor abundance and dynamics are key factors in maintaining memory function and that changes in these parameters are linked to age-dependent short-term memory decline.

LPD-3 as a megaprotein brake for aging and insulin-mTOR signaling in C. elegans.

Insulin-mechanistic target of rapamycin (mTOR) signaling drives anabolic growth during organismal development; its late-life dysregulation contributes to aging and limits lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here, we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin, INS-7, is drastically overproduced from early life and shortens lifespan in lpd-3 mutants. LPD-3 forms a bridge-like tunnel megaprotein to facilitate non-vesicular cellular lipid trafficking. Lipidomic profiling reveals increased hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1. Reducing the abundance of HYL-1, insulin receptor/DAF-2 or mTOR/LET-363, normalizes INS-7 levels and rescues the lifespan of lpd-3 mutants. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age. We propose that LPD-3 acts as a megaprotein brake for organismal aging and that its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Transcriptome analyses describe the consequences of persistent HIF-1 over-activation in Caenorhabditis elegans.

Metazoan animals rely on oxygen for survival, but during normal development and homeostasis, animals are often challenged by hypoxia (low oxygen). In metazoans, many of the critical hypoxia responses are mediated by the evolutionarily conserved hypoxia-inducible transcription factors (HIFs). The stability and activity of HIF complexes are strictly regulated. In the model organism C. elegans, HIF-1 stability and activity are negatively regulated by VHL-1, EGL-9, RHY-1 and SWAN-1. Importantly, C. elegans mutants carrying strong loss-of-function mutations in these genes are viable, and this provides opportunities to interrogate the molecular consequences of persistent HIF-1 over-activation. We find that the genome-wide gene expression patterns are compellingly similar in these mutants, supporting models in which RHY-1, VHL-1 and EGL-9 function in common pathway(s) to regulate HIF-1 activity. These studies illuminate the diversified biological roles played by HIF-1, including metabolism and stress response. Genes regulated by persistent HIF-1 over-activation overlap with genes responsive to pathogens, and they overlap with genes regulated by DAF-16. As crucial stress regulators, HIF-1 and DAF-16 converge on key stress-responsive genes and function synergistically to enable hypoxia survival.

A bioinformatics approach to elucidate conserved genes and pathways in C. elegans as an animal model for cardiovascular research.

Cardiovascular disease (CVD) is a collective term for disorders of the heart and blood vessels. The molecular events and biochemical pathways associated with CVD are difficult to study in clinical settings on patients and in vitro conditions. Animal models play a pivotal and indispensable role in CVD research. Caenorhabditis elegans, a nematode species, has emerged as a prominent experimental organism widely utilized in various biomedical research fields. However, the specific number of CVD-related genes and pathways within the C. elegans genome remains undisclosed to date, limiting its in-depth utilization for investigations. In the present study, we conducted a comprehensive analysis of genes and pathways related to CVD within the genomes of humans and C. elegans through a systematic bioinformatic approach. A total of 1113 genes in C. elegans orthologous to the most significant CVD-related genes in humans were identified, and the GO terms and pathways were compared to study the pathways that are conserved between the two species. In order to infer the functions of CVD-related orthologous genes in C. elegans, a PPI network was constructed. Orthologous gene PPI network analysis results reveal the hubs and important KRs: pmk-1, daf-21, gpb-1, crh-1, enpl-1, eef-1G, acdh-8, hif-1, pmk-2, and aha-1 in C. elegans. Modules were identified for determining the role of the orthologous genes at various levels in the created network. We also identified 9 commonly enriched pathways between humans and C. elegans linked with CVDs that include autophagy (animal), the ErbB signaling pathway, the FoxO signaling pathway, the MAPK signaling pathway, ABC transporters, the biosynthesis of unsaturated fatty acids, fatty acid metabolism, glutathione metabolism, and metabolic pathways. This study provides the first systematic genomic approach to explore the CVD-associated genes and pathways that are present in C. elegans, supporting the use of C. elegans as a prominent animal model organism for cardiovascular diseases.

LET-767 determines lipid droplet protein targeting and lipid homeostasis.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

Feedforward and feedback mechanisms cooperatively regulate rapid experience-dependent response adaptation in a single thermosensory neuron type.

Sensory adaptation allows neurons to adjust their sensitivity and responses based on recent experience. The mechanisms that mediate continuous adaptation to stimulus history over seconds- to hours-long timescales, and whether these mechanisms can operate within a single sensory neuron type, are unclear. The single pair of AFD thermosensory neurons in Caenorhabditis elegans exhibits experience-dependent plasticity in their temperature response thresholds on both minutes- and hours-long timescales upon a temperature upshift. While long-term response adaptation requires changes in gene expression in AFD, the mechanisms driving rapid response plasticity are unknown. Here, we show that rapid thermosensory response adaptation in AFD is mediated via cGMP and calcium-dependent feedforward and feedback mechanisms operating at the level of primary thermotransduction. We find that either of two thermosensor receptor guanylyl cyclases (rGCs) alone is sufficient to drive rapid adaptation, but that each rGC drives adaptation at different rates. rGC-driven adaptation is mediated in part via phosphorylation of their intracellular domains, and calcium-dependent feedback regulation of basal cGMP levels via a neuronal calcium sensor protein. In turn, cGMP levels feedforward via cGMP-dependent protein kinases to phosphorylate a specific subunit of the cGMP-gated thermotransduction channel to further regulate rapid adaptation. Our results identify multiple molecular pathways that act in AFD to ensure rapid adaptation to a temperature change and indicate that the deployment of both transcriptional and nontranscriptional mechanisms within a single sensory neuron type can contribute to continuous sensory adaptation.

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance.

Feeding is an essential biological process for an organism's growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans (C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Optimization of RNAi efficiency in PVD neuron of C. elegans.

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to its elaborate and stereotype dendrite morphology. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to knocking down genes in nervous system. In order to optimize the RNAi conditions for PVD neuron, we fed the worm strains with E. coli HT115 bacteria expressing dsRNA against mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the available sensitive backgrounds including the one expresses sid-1 under unc-119 promoter, although resulted in reduction of dendrite branching, the phenotypes were significantly modest compared to the respective loss of function mutants. In order to enhance RNAi in PVD neurons, we generated a strain that expressed sid-1 under the promoter mec-3, which exhibits strong expression in PVD. When Pmec-3::sid-1 is expressed in either nre-1(-)lin-15b(-) or lin-15b(-) backgrounds, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to the genetic background alone. Moreover, knockdown of genes playing role in dendrite regeneration in the nre-1(-)lin-15b(-), Pmec-3-sid-1[+] background resulted in significant reduction in dendrite regeneration following laser injury. The extent of dendrite regrowth due to the RNAi of aff-1 or ced-10 in our optimized strain was comparable to that of aff-1 and ced-10 mutants. Essentially, our strain expressing sid-1 in PVD neuron, provides an RNAi optimized platform for high throughput screening of genes involved in PVD development, maintenance and regeneration.

The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans.

SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and modulate longevity in C. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. HCF-1 localization at chromatin is largely dependent on functional SET-26, whereas SET-26 is only minorly affected by loss of HCF-1, suggesting that SET-26 could recruit HCF-1 to chromatin. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.