Circadian autophagy drives iTRF-mediated longevity.

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.

FLIRT: fast local infrared thermogenetics for subcellular control of protein function.

FLIRT (fast local infrared thermogenetics) is a microscopy-based technology to locally and reversibly manipulate protein function while simultaneously monitoring the effects in vivo. FLIRT locally inactivates fast-acting temperature-sensitive mutant proteins. We demonstrate that FLIRT can control temperature-sensitive proteins required for cell division, Delta-Notch cell fate signaling, and germline structure in Caenorhabditis elegans with cell-specific and even subcellular precision.

A bidirectional relationship between sleep and oxidative stress in Drosophila.

Although sleep appears to be broadly conserved in animals, the physiological functions of sleep remain unclear. In this study, we sought to identify a physiological defect common to a diverse group of short-sleeping Drosophila mutants, which might provide insight into the function and regulation of sleep. We found that these short-sleeping mutants share a common phenotype of sensitivity to acute oxidative stress, exhibiting shorter survival times than controls. We further showed that increasing sleep in wild-type flies using genetic or pharmacological approaches increases survival after oxidative challenge. Moreover, reducing oxidative stress in the neurons of wild-type flies by overexpression of antioxidant genes reduces the amount of sleep. Together, these results support the hypothesis that a key function of sleep is to defend against oxidative stress and also point to a reciprocal role for reactive oxygen species (ROS) in neurons in the regulation of sleep.

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7 prevents chromosome segregation and cytokinesis defects in oocytes.

In most species, oocytes lack centrosomes. Accurate meiotic spindle assembly and chromosome segregation - essential to prevent miscarriage or developmental defects - thus occur through atypical mechanisms that are not well characterized. Using quantitative in vitro and in vivo functional assays in the C. elegans oocyte, we provide novel evidence that the kinesin-13 KLP-7 promotes destabilization of the whole cellular microtubule network. By counteracting ectopic microtubule assembly and disorganization of the microtubule network, this function is strictly required for spindle organization, chromosome segregation and cytokinesis in meiotic cells. Strikingly, when centrosome activity was experimentally reduced, the absence of KLP-7 or the mammalian kinesin-13 protein MCAK (KIF2C) also resulted in ectopic microtubule asters during mitosis in C. elegans zygotes or HeLa cells, respectively. Our results highlight the general function of kinesin-13 microtubule depolymerases in preventing ectopic, spontaneous microtubule assembly when centrosome activity is defective or absent, which would otherwise lead to spindle microtubule disorganization and aneuploidy.

Patterning, regulation, and role of FoxO/DAF-16 in the early embryo.

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.

Germ fate determinants protect germ precursor cell division by reducing septin and anillin levels at the cell division plane.

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 formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell 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 septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

Neuronal knockdown of Cullin3 as a Drosophila model of autism spectrum disorder.

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.

An unconventional TOG domain is required for CLASP localization.

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/LL5ß.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.

Kinetochore component function in C. elegans oocytes revealed by 4D tracking of holocentric chromosomes.

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.

Synergistic stabilization of microtubules by BUB-1, HCP-1, and CLS-2 controls microtubule pausing and meiotic spindle assembly.

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.

Drosophila mutants lacking the glial neurotransmitter-modifying enzyme Ebony exhibit low neurotransmitter levels and altered behavior.

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.

Germ fate determinants protect germ precursor cell division by restricting septin and anillin levels at the division plane.

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.

Control of nuclear centration in the C. elegans zygote by receptor-independent Galpha signaling and myosin II.

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus-centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Galpha proteins GOA-1/GPA-16. In centration- defective let-99(-) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Galpha signaling and actomyosin. During wild-type centration, NMY-2-GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16-depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(-) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Galpha signaling and LET-99 control centration by regulating polarized actomyosin contraction.

Functional midbody assembly in the absence of a central spindle.

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.

Oligodendroglial macroautophagy is essential for myelin sheath turnover to prevent neurodegeneration and death.

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.

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells.

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Determining the position of the cell division plane.

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.

Circadian regulation of mitochondrial uncoupling and lifespan.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.

BACKGROUND: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. RESULTS: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. CONCLUSIONS: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.