Biology subjects

Mair, W. B.

Publications and source records attributed to Mair, W. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Redox activation of ATG5 licenses autophagy upon nutrient restriction

Dietary restriction (DR) protects against metabolic disease, extends lifespan, and is associated with remodeling of tissue reactive oxygen species (ROS). ROS control biological adaptation through reversible oxidation of protein cysteines, yet the targets of DR-initiated redox signaling are unknown. Here we generate OxiDR, a tissue-resolved atlas of the cysteine redox proteome that quantifies oxidation state under DR. Rather than oxidizing the proteome broadly, DR selectively targets a high-amplitude set of cysteines in a tissue-specific manner, allowing systematic classification of biological processes subject to DR-mediated redox regulation. Among the cysteines most highly oxidized upon DR is Cys19 of the core autophagy protein ATG5. We show oxidation of Cys19 is required for ATG5-mediated autophagosome formation and for autophagy triggered by nutrient restriction in human cells and mice. Reversible oxidation of this cysteine promotes ATG5 binding to ATG10, thus forming the ATG5-ATG12 conjugate that lipidates LC3B/ATG8 and matures the autophagosome. In mice, loss of this redox switch prevents effective initiation of autophagy upon nutrient restriction, resulting in gross tissue pathology and rapid onset of mortality. The autophagic response to nutrient restriction is thus gated by oxidation of a single cysteine.

cell biology↗

bioRxiv2025

Regulation of the Rheb GTPase via GATOR1 Complex

mTORC1 coordinates cellular growth and metabolism by integrating inputs from both amino acids and growth factors, and its activation requires two upstream branches involving the Rag GTPases and the Rheb GTPase. These branches are regulated by distinct GAP complexes: GATOR1 (Depdc5-Nprl2-Nprl3) inhibits RagA/B, and TSC (TSC1-TSC2-TBC1D7) inhibits Rheb. Despite the prevailing view that these pathways converge only at mTORC1 itself, several observations suggest upstream crosstalk. This gap is especially striking in organisms like C. elegans and S. cerevisiae that lack the TSC complex yet maintain fully responsive mTORC1 signaling. How these inputs are dynamically coordinated under complex physiological conditions and in organisms lacking the key components remain unknown. We performed unbiased quantitative proteomics in C. elegans and identified the GATOR1 complex as a previously unrecognized RHEB-1 (C. elegans ortholog of Rheb) interactor. Through biochemical validation in human cells, we show that nucleotide-free Rheb associates with the Nprl2-Nprl3 subunits of GATOR1, whereas GTP-bound or membrane-detached Rheb mutants fail to bind. Nutrient stress, but not direct pharmacologic inhibition of mTORC1, robustly induced this interaction. In TSC2-null cells, where Rheb is constitutively GTP-loaded, Rheb-Nprl2/3 binding was strongly diminished and was restored by expressing the nucleotide-free RhebS20N mutant, demonstrating that Rhebs nucleotide state governs this interaction. Pulldown assays confirmed that the Nprl2/3 heterodimer is sufficient for binding nucleotide-free Rheb. Structural modeling using AlphaFold3 consistently positioned Rheb at a conserved site on Nprl3 distinct from the RagA/B GAP-active surface of Nprl2, supporting a non-catalytic mode of association. Together, these findings identify a conserved, nutrient-regulated physical interaction between Rheb and the Nprl2/3 subunits of GATOR1, revealing a previously unrecognized point of convergence between the growth factor and amino acid branches of the mTORC1 pathway. This model provides a direct molecular link between the Rag and Rheb branches, furthering our understanding of how nutrient stress fine-tunes mTORC1 signaling.

biochemistry↗

bioRxiv2025

Nutrient-Responsive Formation of Mitochondrial-Derived Structures in Caenorhabditis elegans

Mitochondrial morphology is dynamically regulated through remodeling processes essential for maintaining mitochondrial function and ensuring cellular and metabolic homeostasis. While classical models of mitochondrial dynamics center on cycles of fragmentation and elongation, emerging evidence highlights additional membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and mitochondrial-derived compartments (MDCs). These mitochondrial-derived structures, however, have been predominantly characterized in cultured cells and unicellular organisms, leaving their relevance in multicellular systems largely unexplored. Here, we identify a previously uncharacterized class of mitochondrial-derived structures in Caenorhabditis elegans muscle cells that are induced in response to intermittent fasting. We show that these structures appear specifically during the refeeding phase-- coinciding with mitochondrial elongation --and are absent during fasting. Consistent with MDCs, the structures, approximately 1 {micro}m in size, are enriched in outer mitochondrial membrane markers such as TOMM-20aa1-49 and TOMM-70, but notably lack components of the inner mitochondrial membrane. Their formation requires the microtubule-associated MIRO-1/2 proteins, and their size is modulated by the mitochondrial dynamics machinery. Together, our findings reveal a nutritionally regulated mitochondrial remodeling event in C. elegans muscle that may play a role in mitochondrial quality control and adaptation to metabolic cues.

cell biology↗

bioRxiv2025

Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging

Aging results in a loss of metabolic flexibility during fasting, characterized by the inability to acutely switch between metabolic substrates and reduced lipid mobilization. However, the drivers of these effects intracellularly remain unclear. Here, in Caenorhabditis elegans, we show that loss of coordinated inter-organelle dynamics causally initiates metabolic inflexibility with age. In young animals, peroxisomes emerge as the priming orchestrators of the fasting response, simultaneously governing lipid droplets (LDs) utilization and mitochondrial bioenergetics. With age, peroxisomal priming is lost, leading to mitochondrial fragmentation and impaired dynamic nutrient responses during fasting. Notably, dietary restriction (DR) exerts a rejuvenating effect on peroxisomal function, thereby preserving mitochondrial integrity and promoting longevity. Our study uncovers the expansive network of organelles enabling lipid mobilization during youth, providing critical context to the poorly understood role of peroxisomes in actively maintaining organelle homeodynamics and metabolic flexibility throughout the aging process.

cell biology↗

bioRxiv2024

Novel Imaging Tools to Study Mitochondrial Dynamics in Caenorhabditis elegans

Mitochondria exhibit a close interplay between their structure and function. Understanding this intricate relationship requires advanced imaging techniques that can capture the dynamic nature of mitochondria and their impact on cellular processes. However, much of the work on mitochondrial dynamics has been done in single celled organisms or in vitro cell culture. Here, we introduce novel genetic tools for live imaging of mitochondrial networks in the nematode Caenorhabditis elegans, addressing a pressing need for advanced techniques in studying organelle dynamics within live intact multicellular organisms. Through a comprehensive analysis, we directly compare our tools with existing methods, demonstrating their advantages for visualizing mitochondrial morphology and contrasting their impact on organismal physiology. We reveal limitations of conventional techniques, while showcasing the utility and versatility of our approaches, including endogenous CRISPR tags and ectopic labeling. By providing a guide for selecting the most suitable tools based on experimental goals, our work advances mitochondrial research in C. elegans and enhances the strategic integration of diverse imaging modalities for a holistic understanding of organelle dynamics in living organisms.

cell biology↗

bioRxiv2023

Transcriptomic reprogramming screen identifies SRSF1 as rejuvenation factor

Aging is a complex process that manifests through the time-dependent functional decline of a biological system. Age-related changes in epigenetic and transcriptomic profiles have been successfully used to measure the aging process1,2. Moreover, modulating gene regulatory networks through interventions such as the induction of the Yamanaka factors has been shown to reverse aging signatures and improve cell function3,4. However, this intervention has safety and efficacy limitations for in vivo rejuvenation5,6, underscoring the need for identifying novel age reversal factors. Here, we discovered SRSF1 as a new rejuvenation factor that can improve cellular function in vitro and in vivo. Using a cDNA overexpression screen with a transcriptomic readout we identified that SRSF1 induction reprograms the cell transcriptome towards a younger state. Furthermore, we observed beneficial changes in senescence, proteasome function, collagen production, and ROS stress upon SRSF1 overexpression. Lastly, we showed that SRSF1 can improve wound healing in vitro and in vivo and is linked to organismal longevity. Our study provides a proof of concept for using transcriptomic reprogramming screens in the discovery of age reversal interventions and identifies SRSF1 as a promising target for cellular rejuvenation.

genomics↗

bioRxiv2022

Ultra-sensitive coupling between organ growth and size by YAP-1 ensures uniform body plan proportions in C. elegans

Imbalance between the growth rate of different organs can amplify to large deviations of their size proportions during development. We show that, for the C. elegans pharynx, such size divergence is prevented by reciprocal coordination of pharyngeal growth with other tissues. Live imaging of hundreds of individuals revealed that small pharynxes grow more rapidly than large pharynxes, catching up in volume during development. Moreover, pharynx-to-body size proportions were robust to even strong tissue-specific inhibition of mTORC1 and insulin signalling. Tissue-specific depletion of these pathways slowed-down the growth of the respective tissue and additionally triggered a systemic growth response that ensured appropriate organ size proportions. By mathematical modelling, we show that the conservation of proportions requires a bi-directional ultra-sensitive coupling of body growth and pharynx size that cannot be explained by a reduction of food uptake alone. Instead, organ growth coordination requires regulation by the mechano-transducing transcriptional co-activator YAP/yap-1. Knock-down of yap-1 makes animals sensitive to tissue-specific inhibition mTORC1 inhibition, causing a disproportionate pharynx and developmental arrest. Our data suggests that mechano-transduction tightly coordinates organ growth during C. elegans development to ensure the uniformity of body plan proportions among individuals.

developmental biology↗

bioRxiv2022

CRTC-1 Balances Histone Trimethylation and Acetylation to Promote Longevity

Loss of function during ageing is accompanied by transcriptional drift, altering gene expression and contributing to a variety of age-related diseases. CREB-regulated transcriptional coactivators (CRTCs) have emerged as key regulators of gene expression that might be targeted to promote longevity. Here, we define the role of the Caenorhabditis elegans CRTC-1 in the epigenetic regulation of longevity. Endogenous CRTC-1 binds chromatin factors, including components of the COMPASS complex, which trimethylates lysine 4 on histone H3 (H3K4me3). CRISPR editing of endogenous CRTC-1 reveals that the CREB-binding domain in neurons is specifically required for H3K4me3-dependent longevity. However, this effect is independent of CREB but instead acts via the transcription factor AP-1. Strikingly, CRTC-1 also mediates global histone acetylation levels, and this acetylation is essential for H3K4me3-dependent longevity. Indeed, overexpression of an acetyltransferase enzyme is sufficient to promote longevity in wild-type worms. CRTCs, therefore, link energetics to longevity by critically fine-tuning histone acetylation and methylation to promote healthy ageing.

genetics↗

bioRxiv2022

Confirming the Pro-longevity Effects of H3K4me3-deficient set-2 Mutants in Extending Lifespan in C. elegans

The COMPASS chromatin complex, which trimethylates lysine 4 on histone H3 (H3K4me3), regulates lifespan in Caenorhabditis elegans. Knockdown or partial loss-of-function of SET-2, a member of the COMPASS complex, extends the lifespan of worms. However, recent observations suggested that full loss of SET-2 methyltransferase activity via deletion of its active site reduces lifespan, indicating the degree of COMPASS inhibition may be critical to its effects on aging. To further explore these inconsistencies, we examined set-2 longevity across a range of interventions from weak to full inhibition. Via CRISPR/Cas9 genome-editing, we made two new set-2 mutants, a new genocopy of the most commonly used set-2(ok952) allele for mild set-2 inhibition, and a full set-2 genomic deletion. We found that both new strains (partial and null) and RNAi show a lifespan extension in C. elegans fed HT115 E. coli. However, neither mutant was long-lived in C. elegans fed OP50-1 E. coli. These data confirm that the previous lifespan extension observed in set-2(ok952) mutants was indeed the result of set-2 inhibition (and not a secondary linked mutation generated in the original strain). These data also indicate that a diet-dependent mechanism might contribute to the regulation of lifespan under H3K4me3 deficiency and highlight how COMPASS-mediated longevity involves a complex interaction between chromatin state and environment.

genetics↗

bioRxiv2021

A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans.

Translating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types. Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome. Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging.

genetics↗

bioRxiv2021

Efficacy of longevity interventions in C. elegans is determined by early life activity of RNA splicing factors

Geroscience aims to target the aging process to extend healthspan. However, even isogenic individuals show heterogeneity in natural aging rate and responsiveness to pro-longevity interventions, limiting translational potential. Using in vivo mini gene reporters in isogenic C. elegans, we show that alternative splicing of mRNAs related to lipid metabolism in young animals is coupled to subsequent life expectancy. Further, activity of RNA splicing factors REPO-1 and SFA-1 early in life modulates effectiveness of specific longevity interventions via POD-2/ACC1 and regulation of lipid utilization. In addition, early inhibition of REPO-1 renders animals refractory to late onset suppression of the TORC1 pathway. Together these data suggest that activity of RNA splicing factors and the metabolic landscape early in life can modulate responsiveness to longevity interventions and may explain variance in efficacy between individuals. One Sentence SummaryEfficacy of pro-longevity interventions in C. elegans is determined by the activity of splicing factors and the lipid metabolic landscape early in the life of the individual.

genetics↗

bioRxiv2021

Neuronal mTORC1 inhibition promotes longevity without suppressing anabolic growth and reproduction in C. elegans

One of the most robust and reproducible methods to prolong lifespan in a variety of organisms is inhibition of the mTORC1 (mechanistic target of rapamycin complex 1) pathway. mTORC1 is a metabolic sensor that promotes anabolic growth when nutrients are abundant. Inhibition of mTORC1 extends lifespan, but also frequently has other effects such as stunted growth, slowed development, reduced fertility, and disrupted metabolism. It has long been assumed that suppression of anabolism and resulting phenotypes such as impaired growth and reproduction may be causal to mTORC1 longevity, but this hypothesis has not been directly tested. RAGA-1 is an upstream activator of TORC1. Previous work from our lab using a C. elegans model of mTORC1 longevity, the long-lived raga-1 null mutant, found that the presence of raga-1 only in the neurons suppresses longevity of the null mutant. Here, we use the auxin-inducible degradation (AID) system to test whether neuronal mTORC1 inhibition is sufficient for longevity, and whether any changes in lifespan are also linked to stunted growth or fertility. We find that life-long AID of RAGA-1 either in all somatic tissue or only in the neurons of C. elegans is sufficient to extend lifespan. We also find that AID of RAGA-1 or LET-363/mTOR beginning at day 1 of adulthood extends lifespan to a similar extent. Unlike somatic degradation of RAGA-1, neuronal degradation of RAGA-1 doesnt impair growth, slow development, or decrease the reproductive capacity of the worms. Lastly, while AID of LET-363/mTOR in all somatic cells shortens lifespan, neuronal AID of LET-363/mTOR slows aging. This work demonstrates that targeting mTORC1 specifically in the neurons uncouples longevity from growth and reproductive impairments, challenging previously held ideas about the mechanisms of mTORC1 longevity and elucidating the promise of tissue-specific aging therapeutics.

genetics↗

bioRxiv2020

AMPK Modulates Associative Learning via Neuronal Mitochondrial Fusion in C. elegans

Loss of metabolic homeostasis is one of the hallmarks of the aging process that might contribute to pathogenesis by creating a permissive landscape over which neurodegenerative diseases can take hold. AMPK, a conserved energy sensor, extends lifespan and is protective in some neurodegenerative models. AMPK regulates mitochondrial homeostasis and morphology, however, whether mitochondrial regulation causally links AMPK to protection against loss of neuronal function with aging and diseases remains unclear. Here we use an associative learning protocol in C. elegans as a readout of neuronal function and show that AMPK activation enhances associative learning and prevents age-related loss of learning capacity. AMPK promotes neuronal mitochondrial fusion and mitochondrial fragmentation via fzo-1 deletion blocks AMPKs effects on associative learning. Restoring mitochondrial fusion capacity specifically in the neurons rescued learning capacity downstream of AMPK. Finally, AMPK activation rescues neuronal A{beta}1-42 induced loss of associative learning. Overall, our results suggest that targeting neuronal metabolic flexibility may be a viable therapeutic option to restore neuronal function in the context of aging and neurodegenerative diseases.

neuroscience↗

bioRxiv2020

ATF-4 and hydrogen sulfide signalling mediate longevity from inhibition of translation or mTORC1

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

physiology↗

bioRxiv2020

FLN-1/Filamin is required to anchor the actomyosin cytoskeleton and for global organization of sub-cellular organelles in a contractile tissue

Actomyosin networks are organized in space, direction, size, and connectivity to produce coordinated contractions across cells. We use the C. elegans spermatheca, a tube composed of contractile myoepithelial cells, to study how actomyosin structures are organized. FLN-1/filamin is required for the formation and stabilization of a regular array of parallel, contractile, actomyosin fibers in this tissue. Loss of fln-1 results in the detachment of actin fibers from the basal surface, which then accumulate along the cell junctions and are stabilized by spectrin. In addition, actin and myosin are captured at the nucleus by the linker of nucleoskeleton and cytoskeleton complex (LINC) complex, where they form large foci. Nuclear positioning and morphology, distribution of the endoplasmic reticulum and the mitochondrial network are also disrupted. These results demonstrate that filamin is required to prevent large actin bundle formation and detachment, to prevent excess nuclear localization of actin and myosin, and to ensure correct positioning of organelles.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsFRAPfluorescence recovery after photobleachingGFPgreen fluorescent proteinKASHKlarsicht/ANC-1/Syne homologyLINClinker of nucleoskeleton and cytoskeletonmoeABDmoesin actin binding domainRNAiRNA interferencesp-utspermatheca-uterineSUNSad1/UNC-84WTwild typeView Full Text

cell biology↗

bioRxiv2020

Atf-6 regulates lifespan through ER-mitochondrial calcium homeostasis

Functional crosstalk between organelles is critical for maintaining cellular homeostasis. Individually, dysfunction of both endoplasmic reticulum (ER) and mitochondria have been linked to cellular and organismal aging, but little is known about how mechanisms of inter-organelle communication might be targeted to extended longevity. The metazoan unfolded protein response (UPR) maintains ER health through a variety of mechanisms beyond its canonical role in proteostasis, including calcium storage and lipid metabolism. Here we provide evidence that in C. elegans, inhibition of the conserved UPR mediator, activating transcription factor (atf)-6 increases lifespan via modulation of calcium homeostasis and signaling to the mitochondria. Loss of atf-6 confers long life via downregulation of the ER calcium buffering protein, calreticulin. Function of the ER calcium release channel, the inositol triphosphate receptor (IP3R/itr-1), is required for atf-6 mutant longevity while a gain-of-function IP3R/itr-1 mutation is sufficient to extend lifespan. IP3R dysfunction leads to altered mitochondrial behavior and hyperfused morphology, which is sufficient to suppress long life in atf-6 mutants. Highlighting a novel and direct role for this inter-organelle coordination of calcium in longevity, the mitochondrial calcium import channel, mcu-1, is also required for atf-6 mutant longevity. Altogether this study reveals the importance of organellar coordination of calcium handling in determining the quality of aging, and highlights calcium homeostasis as a critical output for the UPR and atf-6 in particular.

cell biology↗

bioRxiv2019

Mitochondrial translation and dynamics synergistically extend lifespan in C. elegans through HLH-30

Mitochondrial form and function, such as translation, are closely interlinked in homeostasis and aging. Inhibiting mitochondrial translation is known to increase lifespan in C. elegans, which is accompanied by a fragmented mitochondrial network. However, the causality between mitochondrial translation and morphology in longevity remains uncharacterized. Here, we show in C. elegans that disrupting mitochondrial network homeostasis by either blocking fission or fusion synergizes with the reduced mitochondrial translation to substantially prolong lifespan and stimulate stress response such as the mitochondrial unfolded protein response, UPRMT. Conversely, immobilizing the mitochondrial network through a simultaneous abrogation of fission and fusion reverses the lifespan increase induced by mitochondrial translation inhibition. Furthermore, we find that the synergistic effect of inhibiting both mitochondrial translation and dynamics on lifespan, despite stimulating UPRMT, does not require it. Instead, this lifespan-extending synergy is exclusively dependent on the lysosome biogenesis and autophagy transcription factor HLH-30/TFEB. Altogether, our study reveals the mechanistic connections between mitochondrial translation and dynamics in regulating longevity. SUMMARYMitochondrial form and function are intimately intertwined. Liu et al. find the synergistic effect of inhibiting both mitochondrial translation and dynamics on lifespan. This synergy is dependent on the induction of lysosome biogenesis through the nuclear localization of HLH-30.

cell biology↗

bioRxiv2019

Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans

Target of rapamycin complex 1 (TORC1) and AMP-activated protein kinase (AMPK) antagonistically modulate metabolism and aging. However, how they coordinate to determine longevity and if they act via separable mechanisms is unclear. Here, we show that neuronal AMPK is essential for lifespan extension from TORC1 inhibition, and that TORC1 suppression increases lifespan cell non autonomously via distinct mechanisms from global AMPK activation. Lifespan extension by null mutations in genes encoding raga-1 (RagA) or rsks-1 (S6K) is fully suppressed by neuronal-specific rescues. Loss of RAGA-1 increases lifespan via maintaining mitochondrial fusion. Neuronal RAGA-1 abrogation of raga-1 mutant longevity requires UNC-64/syntaxin, and promotes mitochondrial fission cell nonautonomously. Finally, deleting the mitochondrial fission factor DRP-1 renders the animal refractory to the pro-aging effects of neuronal RAGA-1. Our results highlight a new role for neuronal TORC1 in cell nonautonomous regulation of longevity, and suggest TORC1 in the central nervous system might be targeted to promote healthy aging.

genetics↗