Biology subjects

Edwards, H.

Publications and source records attributed to Edwards, H..

16 recordsLinked to original sources

bioRxiv2026

Cas1 epistasis tunes conformational coupling to enhance CRISPR adaptation

CRISPR adaptation requires Cas1-Cas2 to capture prespacers, undergo conformational rearrangement and catalyse integration, but how these steps are coupled remains unclear. Using nine hyperactive Escherichia coli Cas1 substitutions as perturbational probes, we identified a prespacer-coupling module intersecting an interior conformational-coupling module. Single substitutions increased adaptation up to sixfold, whereas combinatorial reassortment generated a genotype with 103-fold greater activity than wild type. Genotype-network analysis and quantitative reconstruction revealed strong background-dependent epistasis: the same substitution could enhance activity in one genotype but impair it in another, and high activity emerged only from compatible combinations spanning both modules. These findings indicate that Cas1-Cas2 activity is constrained by compatibility among changes distributed across the Cas1 dimer, rather than by optimization of individual catalytic or DNA-binding interactions. We propose that coordinated tuning of prespacer engagement and conformational coupling governs Cas1-Cas2 activity during CRISPR adaptation.

biochemistry↗

bioRxiv2026

RecA status determines SOS- and RecBCD-dependent outcomes in CRISPR-Cas adaptation

CRISPR-Cas immunity depends on integrating DNA fragments from invading elements, yet how this process is tuned by host physiology remains poorly understood. Here, we investigate the relationship between CRISPR adaptation and the bacterial SOS DNA damage response in Escherichia coli using a highly sensitive colony-based adaptation assay. Blocking SOS with a lexA3 allele suppresses adaptation, while paradoxically, deleting recA enhances it. These findings indicate that RecA contributes positively to adaptation indirectly, through LexA cleavage and induction of SOS-regulated functions, while complete loss of RecA is associated with a RecBCD-dependent DNA-processing state that favours successful adaptation. The RecA inhibitors RecX and PsiB did not phenocopy {triangleup}recA, indicating that inhibitor-mediated perturbation of RecA in RecA-proficient cells and complete loss of RecA have different consequences for adaptation. Genome-wide maps of recovered spacers show that RecA and LexA reshape the relative distribution of chromosomal and plasmid-derived spacers, linking CRISPR adaptation to replication and DNA repair dynamics. Although Cas1-Cas2 catalyses spacer integration autonomously in vitro, our findings suggest that successful adaptation in vivo emerges from interactions between spacer acquisition, DNA repair, and bacterial stress physiology, embedding CRISPR adaptation within cellular networks that balance immune protection with the risk of autoimmunity.

microbiology↗

bioRxiv2026

Metabolic control of mitochondrial plasticity and extracellular vesicle biology drives Cryptococcus neoformans virulence

Metabolic adaptation to nutrient stress is a key but poorly understood driver of fungal virulence. Here, we show that a dominant East Asian lineage of Cryptococcus neoformans (VNIa-5), which disproportionately infects immunocompetent hosts, has undergone lineage-specific rewiring of glucose-responsive stress pathways. Integrating population genomics, transcriptomics, and experimental infection models, we demonstrate that VNIa-5s clinical dominance is not explained by environmental prevalence. Instead, selective activation of Snf1 signalling links glucose limitation to mitochondrial tubularisation, extracellular vesicle remodelling, and enhanced melanization. Under low-glucose conditions, VNIa-5 exhibits marked mitochondrial plasticity and extracellular vesicle compositional shifts resembling hypervirulent outbreak lineages of Cryptococcus gattii. Following experimental induction of dormancy, VNIa-5 shows significantly increased virulence in vivo compared with the closely related but clinically rare VNIa-31 subclade, with host survival tightly correlated with mitochondrial morphology. These findings identify metabolic stress integration as a central mechanism shaping cryptococcal virulence and disease in immunocompetent human hosts.

microbiology↗

bioRxiv2026

Quantification of Aspergillus nidulans Actin Dynamics during Early Growth and Septum Formation

Filamentous fungi have complex, three-dimensional growth patterns and a non-adherent nature, which can present challenges for live-cell imaging for quantitative assessment of dynamic cellular processes. To address these challenges, a live-cell imaging system has been modified to constrain the model fungus Aspergillus nidulans to growth in a single focal plane. This enables high-resolution time-lapse imaging of actin dynamics throughout development using a Lifeact actin marker. This system was used to perform kymographic analysis to quantify actin velocity and hyphal extension rates during early hyphal development. Results show two distinct growth phases: germ tube extension (0.58 m/min) and hyphal extension (1.52 m/min). Actin exhibited bi-directional transport along hyphae with biased movement toward the spore body. Actin was also observed re-localizing from hyphal tips to sites of septum formation indicating active redistribution of cytoskeletal resources based on cellular demands. This technological advancement overcomes longstanding limitations in fungal live-cell imaging and provides a new platform for quantitative systems-level analysis of mycelial development, offering new insights into the spatiotemporal coordination of cytoskeletal dynamics during filamentous growth.

cell biology↗

bioRxiv2025

Targeting Troponin I Phosphorylation for Precision Treatment of Diastolic Dysfunction

BackgroundDefective cardiac relaxation (diastolic dysfunction) is prevalent in heart failure, particularly in the context of diabetes, obesity, hypertension, and ageing, and is associated with increased mortality. Yet there is no available treatment that directly targets this abnormal relaxation. Phosphorylation of troponin I by cAMP-dependent protein kinase A (PKA) promotes relaxation, but global PKA activation has widespread, undesirable effects. Here we investigated whether selectively enhancing troponin I phosphorylation could improve relaxation without engaging the broader PKA signalling network. MethodsTo resolve cAMP signalling at subcellular scales, we combined real-time cAMP measurements with FRET-based genetically encoded reporters targeted to defined nanodomains, alongside biochemical and genetic approaches. Protein-protein interaction surfaces were mapped using peptide-array technology, which guided the design of a disruptor peptide to displace specific interactions. The peptides effects on cardiomyocyte function were assessed in vitro using rodent and human cardiomyocytes with biochemical assays, real-time imaging, and work-loop analysis. In vivo effects were evaluated through echocardiography and haemodynamic measurements. ResultsWe identified a previously unrecognized regulatory mechanism in which the cAMP-hydrolyzing enzyme PDE4D9 interacts with troponin I and restricts local cAMP levels within a nanometre-scale domain, thereby selectively controlling troponin I phosphorylation. We found that PDE4D9-troponin I association is markedly increased in cardiac disease in both rodents and humans. A peptide that displaces PDE4D9 from troponin I in a mouse model of heart failure selectively enhances troponin I phosphorylation, accelerates cardiomyocyte relaxation, and prevents diastolic dysfunction, without compromising systolic contractility. ConclusionsThese findings reveal a highly specific regulatory nanodomain governing troponin I phosphorylation and points to a first-in-class, mechanism-based therapeutic strategy with the potential to address a major unmet contributor to heart failure. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe identify a previously unrecognized, nanometre-scale regulatory mechanism in which PDE4D9 binds troponin I to locally suppress cAMP, limit troponin I phosphorylation and attenuate relaxation. C_LIO_LIWe demonstrate that this mechanism becomes maladaptive in disease: PDE4D9-troponin I interaction is markedly upregulated in failing hearts in both rodents and humans, which can mechanistically explain the impaired cardiac relaxation. C_LIO_LIWe show that this interaction is druggable: a rationally designed peptide can selectively displace PDE4D9, restore localized cAMP-PKA signalling, and normalize myocardial relaxation without global cAMP perturbation. C_LIO_LIDisplacement of PDE4D9 from TPNI improves relaxation with no impact on the calcium transient amplitude and without adversely affecting systolic output and cardiac reserve. C_LI What Are the Clinical Implications?O_LIDiastolic dysfunction, a major cause of heart failure with no targeted treatments, may be amenable to therapies that modulate cAMP signalling with subcellular precision rather than global pathway modulation. C_LIO_LISelective disruption of PDE4D9-troponin I binding offers a mechanism-based approach with potential to restore diastolic function while minimizing off-target effects typical of GPCR-directed therapies. C_LIO_LIBy enhancing relaxation while preserving systolic function and cardiac reserve, this approach may overcome limitations of myofilament-targeted therapies currently under evaluation for diastolic dysfunction that exhibit negative inotropic effects. C_LIO_LIMore broadly, this work establishes a platform for targeting localized signalling domains, a concept that may extend beyond cardiovascular disease to other pathological conditions in which dysregulated cAMP contributes to dysfunction. C_LI

cell biology↗

bioRxiv2025

DIA-PASEF Proteomic Profiling Reveals MpkA-Dependent Iron Stress Responses and Siderophore Biosynthesis in Aspergillus nidulans

Filamentous faungi play essential roles in biotechnology as producers of valuable bioproducts and, conversely, as opportunistic pathogens. Aspergillus nidulans is a widely used model organism for fungal genetics and cell biology; however, comprehensive proteomic references for this species remain limited. In this study, we applied a data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) approach to enable efficient and in-depth proteome profiling of A. nidulans. Leveraging ion mobility-based ion cloud information from DDA-PASEF experiments, we developed DIA-PASEF methods that identified 3,904 proteins across biological triplicates grown in rich medium. Compared to prior studies, this represents an increase in protein identifications of more than 140% and was achieved with more than five-fold reduction in analysis time. We employed this newly developed DIA-PASEF methodology to conduct both proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA protein-kinase deficient mutant ({Delta}mpkA). The {Delta}mpkA strain exhibited expression of approximately 500 additional proteins and occupancy of over 1,800 additional phosphosites relative to a control. Differentially expressed and phosphorylated proteins increased by more than an order of magnitude in the {Delta}mpkA mutant across both iron-replete and iron-deplete conditions. Gene Ontology (GO) enrichment analysis revealed broader and distinct biological processes under iron-depleted conditions, highlighting adaptive responses specific to iron limitation and MAPK pathway disruption. This work establishes a high-coverage proteomic resource for A. nidulans and provides novel insights into fungal stress responses and signaling network perturbation. Importantly, high-throughput proteomic profiling reveals that limited iron availability and MAPK pathway disruption increases siderophore biosynthesis.

systems biology↗

bioRxiv2025

Chemical inhibition of asexual development leads to increased ultimate tensile strength in mycelial material.

This study investigates the chemical inhibition of asexual development in Aspergillus nidulans to enhance the mechanical properties of mycelial materials. We hypothesized that suppressing conidiation using the ornithine decarboxylase inhibitor -difluoromethylornithine (DFMO) would increase material strength by inhibiting asexual development, promoting denser hyphal packing. Mycelial materials were grown in DFMO concentrations (0, 0.05, 0.5, and 5 mM), and conidiation and ultimate tensile strength (UTS) were measured. Results showed a dose-dependent reduction in conidiation, with significant decreases at all DFMO levels (P [≤] 0.05). While lower DFMO concentrations (0.05 and 0.5 mM) did not significantly alter UTS, 5 mM DFMO treatment doubled the materials tensile strength compared to controls (P [≤] 0.05). Scanning electron microscopy confirmed reduced developmental structures in DFMO-treated samples, supporting the hypothesis. The non-linear relationship between conidiation suppression and strength improvement suggests additional mechanisms, such as hyphal morphology or cell wall changes, may contribute. These findings demonstrate that chemical modulation of fungal development can rationally tune mycelial material properties, offering a systematic approach for biomaterial engineering.

bioengineering↗

bioRxiv2025

The impact of fungal developmental structures on mechanical properties of mycelial materials.

This study explores how suppressing asexual development in Aspergillus nidulans enhances the mechanical properties of mycelial materials. Using four aconidial mutants ({Delta}brlA, {Delta}flbA, {Delta}fluG, and fadAG42R) that lack asexual development and a control strain (A28) that undergoes typical asexual development, we found that the absence of asexual development significantly improves mechanical strength. All mutants exhibited higher ultimate tensile strength (UTS) than the control, with {Delta}fluG and {Delta}brlA (fluffy nonsporulating, FNS phenotype) showing the highest UTS. Additionally, fadAG42R and {Delta}flbA (fluffy autolytic dominant, FAD phenotype) demonstrated significantly higher strain at failure (SF), linked to increased autolysis and lower dry cell mass compared to the control and FNS mutants. Solid-state NMR analysis revealed that autolysis in FAD mutants disrupts galactofuranose-related metabolic processes, altering cell wall composition and contributing to higher elasticity. These findings suggest that suppressing asexual development enhances mycelial material strength, while autolysis mechanisms influence elasticity. This research highlights the potential for genetic manipulation in fungi to engineer advanced mycelial-based materials with tailored mechanical properties.

bioengineering↗

bioRxiv2025

Synthetic Generation of Dynamic Omics Data Demonstrates Aspergillus nidulans BrlA Paradoxical Wall Stress Response

We propose a method to generate additional dynamic omics trajectories which could support pathway analysis methods such as enrichment analysis, genetic programming, and machine learning. Using long short-term memory neural networks, we can effectively predict an organisms dynamic response to a stimulus based on an initial dataset with relatively few samples. We present both an in silico proof of principle, based on a model that simulates viral propagation, and an in vitro case study, tracking the dynamics of Aspergillus nidulans BrlA transcript in response to antifungal agent micafungin. Our silico experiment was conducted using a highly noisy dataset with only 25 replicates. This proof of principle shows that this method can operate on biological datasets, which often have high variance and few replicates. Our in silico validation achieved a maximum R2 value of approximately 0.95 on our highly noisy, stochastically simulated data. Our in vitro validation achieves an R2 of 0.71. As with any machine learning application, this method will work better with more data; however, both of our applications attain acceptable validation metrics with very few biological replicates. The in vitro experiments also revealed a novel paradoxical dose-response effect: transcriptional upregulation by Aspergillus nidulans BrlA is highest at an intermediate dose of 10 ng/mL and is reduced at both higher and lower concentrations of micafungin.

genomics↗

bioRxiv2024

Aspergillus nidulans Transcription Factor BrlA is Utilized in a Conidiation-Independent Response to Cell-Wall Stress

Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for both brlA and abaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, the brlA transcript is upregulated while the abaA transcript is not. In addition, when mpkA (last protein kinase in the cell wall integrity signaling pathway) is deleted, brlA expression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in the brlA deletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceThe Aspergillus nidulans transcription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outside A. nidulans. Further, BrlA-mediated conidiation is highly conserved across Aspergillus species, so this new functionality is likely relevant in other Aspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in a brlA deletion mutant. Based on our observation, together with what is known about the brlA gene locus regulation, we identify brlA{beta} as the likely intermediary in function of brlA in the response to cell-wall stress.

cell biology↗

bioRxiv2024

Aspergillus nidulans cell wall integrity kinase, MpkA, impacts cellular phenotypes that alter mycelial-material mechanical properties

Mycelial materials are an emerging, natural material made from filamentous fungi that have the potential to replace unsustainable materials used in numerous commercial applications (e.g., packaging, textiles, construction). Efforts to change the mechanical properties of mycelial-materials have typically involved altering growth medium, processing approaches, or fungal species. Although these efforts have shown varying levels of success, all approaches have shown there is a strong correlation between phenotype (of both fungal mycelia and mycelial materials assembly) and resultant mechanical properties. We hypothesize that genetic means can be used to generate specific fungal phenotypes, leading to mycelial materials with specific mechanical properties. To begin to test this hypothesis, we used a mutant of the model filamentous fungus, Aspergillus nidulans, with a deletion of the last kinase in the cell wall integrity (CWI) signaling pathway, mpkA. We generated one set of mycelial materials from the {Delta}mpkA deletion mutant (A1404), and another from its isogenic parent (A1405; control). When subjected to tensile testing, and compared to material generated from the control, {Delta}mpkA material has similar elastic modulus, but significantly increased ultimate tensile strength, and strain at failure. When subjected to a fragmentation assay (i.e., resistance to shear-stress), the {Delta}mpkA material also had higher relative mechanical strength. To determine possible causes for this behavior, we carried out a comprehensive set of phenotype assessments focused on: three-dimensional structure, hyphal morphology, hyphal growth behaviors, and conidial development. We find, compared to the control, material generated from the {Delta}mpkA mutant manifests significantly less development, a modified cell wall composition, larger diameter hyphae, more total biomass, higher water capacity and more densely packed material, which all appear to impact the altered mechanical properties.

bioengineering↗

bioRxiv2024

Using flux theory in dynamic omics data sets to identify differentially changing signals using DPoP

Derivative profiling (DP) is a novel approach to identify differential signals from dynamic omics data sets. This approach applies variable step-size differentiation to time dynamic omics data. This work assumes that there is a general omics derivative that is a useful and descriptive feature of dynamic omics experiments. We assert that this omics derivative, or omics flux, is a valuable descriptor that can be used instead of, or with, fold change calculations. The results of derivative profiling are compared to established methods such as Multivariate Adaptive Regression Splines (MARS), significance versus fold change analysis (Volcano), and an adjusted ratio over intensity (M/A) analysis to find that there is a statistically significant similarity between the results. This comparison is repeated for transcriptomic and phosphoproteomic expression profiles previously characterized in Aspergillus nidulans. This method has been packaged in an open-source, GUI-based MATLAB app, the Derivative Profiling omics Package (DPoP). Gene Ontology (GO) term enrichment has been included in the app so that a user can automatically/programmatically describe the over/under-represented GO terms in the derivative profiling results using domain specific knowledge found in their organisms specific GO database file. The advantage of the DPoP analysis is that it is computationally inexpensive, it does not require fold change calculations, it describes both instantaneous as well as overall behavior, and it achieves statistical confidence with signal trajectories of a single bio-replicate over four or more points. While we apply this method to time dynamic transcriptomic and phosphoproteomic datasets, it is a numerically generalizable technique that can be applied to any organism and any field interested in time series data analysis. The app described in this work enables omics researchers with no computer science background to apply derivative profiling to their data sets, while also allowing multidisciplined users to build on the nascent idea of profiling derivatives in omics.

bioinformatics↗

bioRxiv2024

Protein Kinases MpkA and SepH Transduce Crosstalk Between CWI and SIN Pathways to Activate Protective Hyphal Septation Under Echinocandin Cell Wall Stress

This study investigates a previously unreported stress signal transduced as crosstalk between the Cell Wall Integrity (CWI) pathway and the Septation Initiation Network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific; our findings suggest this is due largely to CWI-SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of {beta}-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery, of previously unknown crosstalk between the CWI and SIN pathways, not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also unveil, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. Hence, this work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin resistant species.

microbiology↗

bioRxiv2020

Characterization of Met25 as a Color Associated Genetic Market in Yarrowia lipolytica

Yarrowia lipolytica offers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily with URA3. In this work, we investigate MET25, a locus of sulfide housekeeping within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast. MET25 deficient Y. lipolytica could be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.

microbiology↗

bioRxiv2019

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5-phosphate decarboxylase ([~]96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%-75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3 polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%-83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.

synthetic biology↗

bioRxiv2019

NemaLife: A structured microfluidic culture device optimized for aging studies in crawling C. elegans

Caenorhabditis elegans is a powerful animal model in aging research. Standard longevity assays on agar plates involve the tedious task of picking and transferring animals to prevent younger progeny from contaminating age-synchronized adult populations. Large-scale studies employ progeny-blocking drugs or sterile mutants to avoid progeny contamination, but such manipulations change adult physiology and alter the influence of reproduction on normal aging. Moreover, for some agar growth-based technology platforms, such as automated lifespan machines, reagents such as food or drugs cannot be readily added/removed after initiation of the study. Current microfluidic approaches are well-suited to address these limitations, but in their liquid-based environments animals swim rather than crawl, introducing swim-induced stress in the lifespan analysis. Here we report a simple microfluidic device that we call NemaLife that features: 1) an optimized micropillar arena in which animals can crawl, 2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and 3) ports which allow rapid accessibility to feed the adult-only population and introduce reagents as needed. Culture maintenance and liquid manipulation are performed with simple hand-held syringes to facilitate integration of our technology into general laboratory protocols. Additionally, device geometry and feeding protocols were designed to emulate the body gait, locomotion, and lifespan of animals reared on agar. We validated our approach with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan. Overall, the capacity to generate reliable lifespan and physiological data from the NemaLife chip underscores the potential of this device to accelerate healthspan and lifespan investigations in C. elegans.

bioengineering↗