The gut microbiome and its bacterially derived metabolites are known to affect many aspects of the host organism's health, including metabolism, immune response, intestinal inflammation, oxidative stress, and even lifespan. Because pathological changes in the gut microbiome and these functions are associated with aging, many have hypothesized that we could protect against aging by generating beneficial changes to the gut microbiome. Here, we directed evolution outside of the host ( ex vivo ) and generated a Drosophila gut microbiome resistant to paraquat, a toxin that causes oxidative stress. Compared to a control microbiome, this paraquat-resistant (PQR) microbiome transplanted back into the Drosophila gut endowed the host with multiple health benefits: increased resistance to dietary paraquat, reduced age-related pathologies in the gut, and extended lifespan. We identified the beneficial species of the PQR microbiome as Lactiplantibacillus plantarum and further identified mutations specific to lifespan-extending isolates linked to greater production of acetate. Directly feeding this short-chain fatty acid, acetate, to Drosophila was sufficient to recapitulate an extended lifespan, similar to that induced by gut colonization of PQR bacteria in the gut. These results serve as a proof of principle that increasing the resistance of the microbiome to oxidative stress via directed ex vivo evolution could serve as a therapeutic strategy to protect against aging.

Forkhead box O transcription factors (FoxO/DAF-16 in C. elegans ) play a conserved role in metabolism, aging, and development. Canonically, FoxO/DAF-16 is inhibited by insulin receptor signaling (DAF -2 in C. elegans ) but also can be regulated by other pathways. Indeed, severe loss-of-function daf-2 ; daf-16 double mutants have high embryonic lethality despite low lethality in either single mutant; the cause of this synthetic lethality is not known. We used genetics and quantitative imaging to study DAF-16 in early C. elegans embryos. Before the 8-cell stage, DAF-16 localized uniformly at low levels in all nuclei. In 8-cell to 64-cell embryos, DAF-16 became enriched in only 1-2 cell nuclei, which lineage tracing revealed to be germ lineage cells. This DAF-16 patterning required germ fate determinants, the phospholipid phosphatase PTEN/DAF-18, but was DAF-2 activity- and maternal age-independent. We found that severe loss-of-function daf-16; daf-2 double mutant embryos failed to undergo morphogenesis with major mitotic chromosome segregation defects as early as the 1-cell stage. Together, our results identify a novel germ lineage-specific patterning of DAF-16 and a canonical DAF-2 signaling-independent role for DAF-16 during early embryogenesis.

ABSTRACT While current therapeutics restricting calorie intake, such as GLP-1 agonists, induce fat loss for many people, they are ineffective for others and concerns remain about their long-term effects on health, particularly loss of lean muscle mass. Moreover, many quickly regain fat if they stop treatment. In contrast, time-restricted eating does not restrict calorie intake but instead restricts the time window for eating and prevents obesity in mice and humans. Here we investigated the effects of intermittent T ime- R estricted F eeding (iTRF), which extends lifespan and delays markers of aging, on stored fat in Drosophila . Ten days of iTRF caused significant fat loss relative to ad lib diet, an effect that persisted even after return to ad lib diet. Unlike iTRF-induced lifespan extension, iTRF-induced fat loss did not depend on circadian-regulated autophagy. iTRF treated both diet-induced and genetically induced obesity and significantly reduced lipid droplet size in the fat body (adipose tissue). Instead of causing muscle loss, iTRF increased total and muscle-specific protein levels and enhanced flight performance, suggesting a shift in body composition. We found that iTRF evoked fasting-induced hyperactivity, partially mediated by octopamine, the fly ortholog of the human stress hormone norepinephrine. Ablation of octopaminergic neurons (OANs) prevented iTRF-mediated effects: fat loss, increased protein, and enhanced flight performance. Our results suggest that Drosophila iTRF causes rapid, permanent fat loss and increased muscle function through a “fight or flight” response. Understanding the mechanisms driving differences between Drosophila and human responses to TRE could be critical for identifying effective therapeutic targets for obesity.

Microtubules are dynamic, conserved cytoskeletal filaments that are essential for all eukaryotic cells. Microtubule dynamic properties are primarily characterized by measuring how fast they grow and shrink (growth and shrinkage rates) and how often they switch between assembly and disassembly (catastrophe and rescue frequencies). These four parameters measured for individual filaments can inform on the behavior of the entire microtubule network at the cell level. By comparing microtubule dynamics in Caenorhabditis elegans one-cell embryos using different genetically-encoded fluorescent probes, we observed an unexpected high variability in these parameters. Microtubule dynamics parameters were consistently higher in C. elegans strains expressing a fluorescently labelled microtubule end-binding protein than in strains relying on tubulin labelling, with microtubule growth rates differing by nearly a factor of two between the two conditions. This discrepancy was not limited to C. elegans, as we observed a similar effect in embryos of the tunicate Phallusia mammillata. Despite this, spindle size and assembly timing were only mildly affected. However, embryos expressing labelled end-binding protein exhibited higher frequency of mitotic defects and perturbed embryonic development upon exposure to various stresses such as elevated temperature or a compromised spindle assembly checkpoint. Thus, our work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions. Our findings should also serve as a cautionary note when using tagged end-binding proteins to measure microtubule dynamics.

Chromosome segregation during anaphase occurs through two mechanistically distinct processes: anaphase A, in which chromosomes move toward spindle poles, and anaphase B, in which the anaphase spindle elongates through cortical astral microtubule pulling forces. Caenorhabditis elegans embryos have been thought to rely primarily on anaphase B, with little to no contribution from anaphase A. Here, we uncover a novel anaphase A mechanism in C. elegans embryos, driven by the kinesin-13 KLP-7MCAK and opposed by the kinesin-12 KLP-18. We found that the extent of chromosome segregation during anaphase A is asymmetrically regulated by cell polarity cues and modulated by mechanical tension within the spindle, generated by opposing forces acting on chromosomes and spindle poles. Additionally, we found that the contribution of anaphase A to chromosome segregation increases progressively across early embryonic divisions. These findings uncover an unexpected role for anaphase A in early C. elegans development and reveal a KLP-7MCAK-dependent mechanical coordination between anaphase A- and anaphase B-driven chromosome segregation.

In previous work, we found that short sleep caused sensitivity to oxidative stress; here we set out to characterize the physiological state of a diverse group of chronically short-sleeping mutants during hyperoxia as an acute oxidative stress. Using RNA-sequencing analysis, we found that short-sleeping mutants had a normal transcriptional oxidative stress response relative to controls. In both short-sleeping mutants and controls, hyperoxia led to downregulation of glycolytic genes and upregulation of genes involved in fatty acid metabolism, reminiscent of metabolic shifts during sleep. We hypothesized that short-sleeping mutants may be sensitive to hyperoxia because of defects in metabolism. Consistent with this, short-sleeping mutants were sensitive to starvation. Using metabolomics, we identified a pattern of low levels of long chain fatty acids and lysophospholipids in short-sleeping mutants relative to controls during hyperoxia, suggesting a defect in lipid metabolism. Though short-sleeping mutants did not have common defects in many aspects of lipid metabolism (basal fat stores, usage kinetics during hyperoxia, respiration rates, and cuticular hydrocarbon profiles), they were all sensitive to dehydration, suggesting a general defect in cuticular hydrocarbons, which protect against dehydration. To test the bi-directionality of sleep and lipid metabolism, we tested flies with both diet-induced obesity and genetic obesity. Flies with diet-induced obesity had no sleep or oxidative stress phenotype; in contrast, the lipid metabolic mutant, brummer , slept significantly more than controls but was sensitive to oxidative stress. Previously, all short sleepers tested were sensitive and all long sleepers resistant to oxidative stress. brummer mutants, the first exceptions to this rule, lack a key enzyme required to mobilize fat stores, suggesting that a defect in accessing lipid stores can cause sensitivity to oxidative stress. Taken together, we found that short-sleeping mutants have many phenotypes in common: sensitivity to oxidative stress, starvation, dehydration, and defects in lipid metabolites. These results argue against a specific role for sleep as an antioxidant and suggest the possibility that lipid metabolic defects underlie the sensitivity to oxidative stress of short-sleeping mutants.

Mutations in Cullin-3 (Cul3), a conserved gene encoding a ubiquitin ligase, are strongly associated with autism spectrum disorder (ASD). Here, we characterize ASD-related pathologies caused by neuron-specific Cul3 knockdown in Drosophila. We confirmed that neuronal Cul3 knockdown causes short sleep, paralleling sleep disturbances in ASD. Because sleep defects and ASD are linked to metabolic dysregulation, we tested the starvation response of neuronal Cul3 knockdown flies; they starved faster and had lower triacylglyceride levels than controls, suggesting defects in metabolic homeostasis. ASD is also characterized by increased biomarkers of oxidative stress; we found that neuronal Cul3 knockdown increased sensitivity to hyperoxia, an exogenous oxidative stress. Additional hallmarks of ASD are deficits in social interactions and learning. Using a courtship suppression assay that measures social interactions and memory of prior courtship, we found that neuronal Cul3 knockdown reduced courtship and learning compared to controls. Finally, we found that neuronal Cul3 depletion alters the anatomy of the mushroom body, a brain region required for memory and sleep. Taken together, the ASD-related phenotypes of neuronal Cul3 knockdown flies establish these flies as a genetic model to study molecular and cellular mechanisms underlying ASD pathology, including metabolic and oxidative stress dysregulation and neurodevelopment.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formin cyk-1 (ts) mutant C. elegans embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide without detectable F-actin at the division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septin UNC-59 and anillin ANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into cytokinetic regulation in other cell types, especially in stem cells with high potency.

Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early 2-cell through 8-cell C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.

At the end of cell division, the nuclear envelope reassembles around the decondensing chromosomes. Female meiosis culminates in two consecutive cell divisions of the oocyte, meiosis I and II, which are separated by a brief transition phase known as interkinesis. Due to the absence of chromosome decondensation and the suppression of genome replication during interkinesis, it has been widely assumed that the nuclear envelope does not reassemble between meiosis I and II. By analyzing interkinesis in C. elegans oocytes, we instead show that an atypical structure made of two lipid bilayers, which we termed the interkinetic envelope, surrounds the surface of the segregating chromosomes. The interkinetic envelope shares common features with the nuclear envelope but also exhibits specific characteristics that distinguish it, including its lack of continuity with the endoplasmic reticulum, unique protein composition, assembly mechanism, and function in chromosome segregation. These distinct attributes collectively define the interkinetic envelope as a unique and specialized structure that has been previously overlooked.

During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C. elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28ELYS. Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28ELYS. Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2M18BP1 (or MEL-28ELYS) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2M18BP1 and MEL-28ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2M18BP1 recruits MEL-28ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C. elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2M18BP1 and MEL-28ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin.

Insulin resistance and diabetes are associated with many health issues including higher rates of birth defects and miscarriage during pregnancy. Because insulin resistance and diabetes are both associated with obesity, which also affects fertility, the role of insulin signaling itself in embryo development is not well understood. A key downstream target of the insulin/insulin-like growth factor signaling (IIS) pathway is the forkhead family transcription factor FoxO (DAF-16 in C. elegans ). Here, we used quantitative live imaging to measure the patterning of endogenously tagged FoxO/DAF-16 in the early worm embryo. In 2-4-cell stage embryos, FoxO/DAF-16 initially localized uniformly to all cell nuclei, then became dramatically enriched in germ precursor cell nuclei beginning at the 8-cell stage. This nuclear enrichment in early germ precursor cells required germ fate specification, PI3K (AGE-1)- and PTEN (DAF-18)-mediated phospholipid regulation, and the deubiquitylase USP7 (MATH-33), yet was unexpectedly insulin receptor (DAF-2)- and AKT-independent. Functional analysis revealed that FoxO/DAF-16 acts as a cell cycle pacer for early cleavage divisions-without FoxO/DAF-16 cell cycles were shorter than in controls, especially in germ lineage cells. These results reveal the germ lineage specific patterning, upstream regulation, and cell cycle role for FoxO/DAF-16 during early C. elegans embryogenesis.

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.

Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. Here, we investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster . Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. Indeed, ebony mutants have been described since 1960 as "aggressive mutants," though this behavior has not been quantified. Using automated machine learning-based analyses, we quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, we found that ebony ’s role in these behaviors was specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. We further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent.

Cytoplasmic linker-associated proteins (CLASPs) form a conserved family of microtubule-associated proteins (MAPs) that maintain microtubules in a growing state by promoting rescue while suppressing catastrophe.1 CLASP function involves an ordered array of tumor overexpressed gene (TOG) domains and binding to multiple protein partners via a conserved C-terminal domain (CTD).2,3 In migrating cells, CLASPs concentrate at the cortex near focal adhesions as part of cortical microtubule stabilization complexes (CMSCs), via binding of their CTD to the focal adhesion protein PHLDB2/LL5β.4,5 Cortical CLASPs also stabilize a subset of microtubules, which stimulate focal adhesion turnover and generate a polarized microtubule network toward the leading edge of migrating cells. CLASPs are also recruited to the trans-Golgi network (TGN) via an interaction between their CTD and the Golgin protein GCC185.6 This allows microtubule growth toward the leading edge of migrating cells, which is required for Golgi organization, polarized intracellular transport, and cell motility.7 In dividing cells, CLASPs are essential at kinetochores for efficient chromosome segregation and anaphase spindle integrity.8,9 Both CENP-E and ASTRIN bind and target CLASPs to kinetochores,10,11 although the CLASP domain required for this interaction is not known. Despite its high evolutionary conservation, the CTD remains structurally uncharacterized. Here, we find that the CTD can be structurally modeled as a TOG domain. We identify a surface-exposed and conserved arginine residue essential for CLASP CTD interaction with partner proteins. Together, our results provide a structural mechanism by which the CLASP CTD directs diverse sub-cellular localizations throughout the cell cycle.

During cell division, chromosome segregation is orchestrated by a microtubule-based spindle. Interaction between spindle microtubules and kinetochores is central to the bi-orientation of chromosomes. Initially dynamic to allow spindle assembly and kinetochore attachments, which is essential for chromosome alignment, microtubules are eventually stabilized for efficient segregation of sister chromatids and homologous chromosomes during mitosis and meiosis I, respectively. Therefore, the precise control of microtubule dynamics is of utmost importance during mitosis and meiosis. Here, we study the assembly and role of a kinetochore module, comprised of the kinase BUB-1, the two redundant CENP-F orthologs HCP-1/2, and the CLASP family member CLS-2 (hereafter termed the BHC module), in the control of microtubule dynamics in Caenorhabditis elegans oocytes. Using a combination of in vivo structure-function analyses of BHC components and in vitro microtubule-based assays, we show that BHC components stabilize microtubules, which is essential for meiotic spindle formation and accurate chromosome segregation. Overall, our results show that BUB-1 and HCP-1/2 do not only act as targeting components for CLS-2 at kinetochores, but also synergistically control kinetochore-microtubule dynamics by promoting microtubule pause. Together, our results suggest that BUB-1 and HCP-1/2 actively participate in the control of kinetochore-microtubule dynamics in the context of an intact BHC module to promote spindle assembly and accurate chromosome segregation in meiosis.

During cell division, chromosome congression to the spindle center, their orientation along the spindle long axis and alignment at the metaphase plate depend on interactions between spindle microtubules and kinetochores, and are pre-requisite for chromosome bi-orientation and accurate segregation. How these successive phases are controlled during oocyte meiosis remains elusive. Here we provide 4D live imaging during the first meiotic division in C. elegans oocytes with wild-type or disrupted kinetochore protein function. We show that, unlike in monocentric organisms, holocentric chromosome bi-orientation is not strictly required for accurate chromosome segregation. Instead, we propose a model in which initial kinetochore-localized BHC module (comprised of BUB-1Bub1, HCP-1/2CENP-F and CLS-2CLASP)-dependent pushing acts redundantly with Ndc80 complex-mediated pulling for accurate chromosome segregation in meiosis. In absence of both mechanisms, homologous chromosomes tend to co-segregate in anaphase, especially when initially mis-oriented. Our results highlight how different kinetochore components cooperate to promote accurate holocentric chromosome segregation in oocytes of C. elegans.

Although macroautophagy deficits are implicated across adult-onset neurodegenerative diseases, we understand little about how the discrete, highly evolved cell types of the central nervous system use macroautophagy to maintain homeostasis. One such cell type is the oligodendrocyte, whose myelin sheaths are central for the reliable conduction of action potentials. Using an integrated approach of mouse genetics, live cell imaging, electron microscopy, and biochemistry, we show that mature oligodendrocytes require macroautophagy to degrade cell autonomously their myelin by consolidating cytosolic and transmembrane myelin proteins into an amphisome intermediate prior to degradation. We find that disruption of autophagic myelin turnover leads to changes in myelin sheath structure, ultimately impairing neural function and culminating in an adult-onset progressive motor decline, neurodegeneration, and death. Our model indicates that the continuous and cell-autonomous maintenance of the myelin sheath through macroautophagy is essential, shedding insight into how macroautophagy dysregulation might contribute to neurodegenerative disease pathophysiology.

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F-like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2-dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle-independent midbodies appeared to form via contractile ring constriction-driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1–5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension. Circadian-regulated autophagy contributes to the health benefits of intermittent time-restricted feeding in Drosophila.