Factors affecting de novo formation of a yeast prion

Prions are aggregates of misfolded proteins that have acquired an amyloid-like structure and ability to propagate through recruitment of new proteins. [PSI+], a prion form of eukaryotic release factor Sup35 (eRF1) is widely used as a model for research on prion formation and propagation and in this study [PSI+] is used to explore an effect of three previously identified proteins on de novo prion formation. One mechanism proposed to affect prion formation is direct interaction of Sup35p with its binding partners and search for proteins that interact with Sup35p identified Ppq 1 p, a putative Ser/Thr protein phosphatase (M.F. Tuite and T. von der Haar). Another approach was to identify proteins that function to protect translational apparatus from environmental and . endogenous oxidative damage. and this approach identified two ribosome associated peroxiredoxins, Tsa1 p and Tsa2p (T. Sideri and C.M. Grant). The data presented here shows that the deletion of PPQ1 gene greatly increases the rate of de novo formation of [PSI+] but the mechanism by which loss of Ppq1 p affects [PSI+] formation is not known. Analysis of the distribution of fluorescently-tagged Ppq 1 P showed that the protein co-localises with mitochondria. A further line of evidence linking Ppq 1 P to mitochondria was an observed reduction in respiratory capacity of a ppq1 Δ strain. That exposure to environmental sources of oxidative stress promotes [PSI+] prion formation was previously reported (Tyedmers et al., 2008). Results presented here show that an endogenous source of oxidative stress, brought about by deleting the ribosomally- associated peroxiredoxins (Prx) encoded by genes TSA 1/2 (Trotter et al., 2008; Sideri et al., 2010), also increases the rate of de novo [PSI+]formation. This result provides a direct link between oxidative stress and the eukaryotic release factor Sup35p

Coupling of growth rate and developmental tempo reduces body size heterogeneity in C. elegans.

Animals increase by orders of magnitude in volume during development. Therefore, small variations in growth rates among individuals could amplify to a large heterogeneity in size. By live imaging of C. elegans, we show that amplification of size heterogeneity is prevented by an inverse coupling of the volume growth rate to the duration of larval stages and does not involve strict size thresholds for larval moulting. We perturb this coupling by changing the developmental tempo through manipulation of a transcriptional oscillator that controls the duration of larval development. As predicted by a mathematical model, this perturbation alters the body volume. Model analysis shows that an inverse relation between the period length and the growth rate is an intrinsic property of genetic oscillators and can occur independently of additional complex regulation. This property of genetic oscillators suggests a parsimonious mechanism that counteracts the amplification of size differences among individuals during development

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

mTORC1 (mechanistic target of rapamycin complex 1) is a metabolic sensor that promotes growth when nutrients are abundant. Ubiquitous inhibition of mTORC1 extends lifespan in multiple organisms but also disrupts several anabolic processes resulting in stunted growth, slowed development, reduced fertility, and disrupted metabolism. However, it is unclear if these pleotropic effects of mTORC1 inhibition can be uncoupled from longevity. Here, we utilize the auxin-inducible degradation (AID) system to restrict mTORC1 inhibition to C. elegans neurons. We find that neuron-specific degradation of RAGA-1, an upstream activator of mTORC1, or LET-363, the ortholog of mammalian mTOR, is sufficient to extend lifespan in C. elegans. Unlike raga-1 loss of function genetic mutations or somatic AID of RAGA-1, neuronal AID of RAGA-1 robustly extends lifespan without impairing body size, developmental rate, brood size, or neuronal function. Moreover, while degradation of RAGA-1 in all somatic tissues alters the expression of thousands of genes, demonstrating the widespread effects of mTORC1 inhibition, degradation of RAGA-1 in neurons only results in around 200 differentially expressed genes with a specific enrichment in metabolism and stress response. Notably, our work demonstrates that targeting mTORC1 specifically in the nervous system in C. elegans uncouples longevity from growth and reproductive impairments, and that many canonical effects of low mTORC1 activity are not required to promote healthy aging. These data challenge previously held ideas about the mechanisms of mTORC1 lifespan extension and underscore the potential of promoting longevity by neuron-specific mTORC1 modulation

Factors affecting de novo formation of a yeast prion

Prions are aggregates of misfolded proteins that have acquired an amyloid-like structure and ability to propagate through recruitment of new proteins. [PSI+], a prion form of eukaryotic release factor Sup35 (eRF1) is widely used as a model for research on prion formation and propagation and in this study [PSI+] is used to explore an effect of three previously identified proteins on de novo prion formation. One mechanism proposed to affect prion formation is direct interaction of Sup35p with its binding partners and search for proteins that interact with Sup35p identified Ppq 1 p, a putative Ser/Thr protein phosphatase (M.F. Tuite and T. von der Haar). Another approach was to identify proteins that function to protect translational apparatus from environmental and . endogenous oxidative damage. and this approach identified two ribosome associated peroxiredoxins, Tsa1 p and Tsa2p (T. Sideri and C.M. Grant). The data presented here shows that the deletion of PPQ1 gene greatly increases the rate of de novo formation of [PSI+] but the mechanism by which loss of Ppq1 p affects [PSI+] formation is not known. Analysis of the distribution of fluorescently-tagged Ppq 1 P showed that the protein co-localises with mitochondria. A further line of evidence linking Ppq 1 P to mitochondria was an observed reduction in respiratory capacity of a ppq1 Δ strain. That exposure to environmental sources of oxidative stress promotes [PSI+] prion formation was previously reported (Tyedmers et al., 2008). Results presented here show that an endogenous source of oxidative stress, brought about by deleting the ribosomally- associated peroxiredoxins (Prx) encoded by genes TSA 1/2 (Trotter et al., 2008; Sideri et al., 2010), also increases the rate of de novo [PSI+]formation. This result provides a direct link between oxidative stress and the eukaryotic release factor Sup35p.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ribosome-associated peroxiredoxins suppress oxidative stress–induced de novo formation of the [PSI+] prion in yeast

Peroxiredoxins (Prxs) are ubiquitous antioxidants that protect cells against oxidative stress. We show that the yeast Tsa1/Tsa2 Prxs colocalize to ribosomes and function to protect the Sup35 translation termination factor against oxidative stress–induced formation of its heritable [PSI+] prion conformation. In a tsa1 tsa2 [psi-] [PIN+] strain, the frequency of [PSI+] de novo formation is significantly elevated. The Tsa1/Tsa2 Prxs, like other 2-Cys Prxs, have dual activities as peroxidases and chaperones, and we show that the peroxidase activity is required to suppress spontaneous de novo [PSI+] prion formation. Molecular oxygen is required for [PSI+] prion formation as growth under anaerobic conditions prevents prion formation in the tsa1 tsa2 mutant. Conversely, oxidative stress conditions induced by exposure to hydrogen peroxide elevates the rate of de novo [PSI+] prion formation leading to increased suppression of all three termination codons in the tsa1 tsa2 mutant. Altered translational fidelity in [PSI+] strains may provide a mechanism that promotes genetic variation and phenotypic diversity (True HL, Lindquist SL (2000) Nature 407:477–483). In agreement, we find that prion formation provides yeast cells with an adaptive advantage under oxidative stress conditions, as elimination of the [PSI+] prion from tsa1 tsa2 mutants renders the resulting [psi-] [pin-] cells hypersensitive to hydrogen peroxide. These data support a model in which Prxs function to protect the ribosomal machinery against oxidative damage, but when these systems become overwhelmed, [PSI+] prion formation provides a mechanism for uncovering genetic traits that aid survival during oxidative stress conditions

Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1

Abstract Even slight imbalance between the growth rate of different organs can accumulate to a large deviation from their appropriate size during development. Here, we use live imaging of the pharynx of C. elegans to ask if and how organ size scaling nevertheless remains uniform among individuals. Growth trajectories of hundreds of individuals reveal that pharynxes grow by a near constant volume per larval stage that is independent of their initial size, such that undersized pharynxes catch-up in size during development. Tissue-specific depletion of RAGA-1, an activator of mTOR and growth, shows that maintaining correct pharynx-to-body size proportions involves a bi-directional coupling between pharynx size and body growth. In simulations, this coupling cannot be explained by limitation of food uptake alone, and genetic experiments reveal an involvement of the mechanotransducing transcriptional co-regulator yap-1. Our data suggests that mechanotransduction coordinates pharynx growth with other tissues, ensuring body plan uniformity among individuals

Effects of stereotypic behaviour and chronic mild stress on judgement bias in laboratory mice

Cognitive processes are influenced by underlying affective states, and tests of cognitive bias have recently been developed to assess the valence of affective states in animals. These tests are based on the fact that individuals in a negative affective state interpret ambiguous stimuli more pessimistically than individuals in a more positive state. Using two strains of mice we explored whether unpredictable chronic mild stress (UCMS) can induce a negative judgement bias and whether variation in the expression of stereotypic behaviour is associated with variation in judgement bias. Sixteen female CD-1 and 16 female C57BL/6 mice were trained on a tactile conditional discrimination test with grade of sandpaper as a cue for differential food rewards. Once they had learned the discrimination, half of the mice were subjected to UCMS for three weeks to induce a negative affective state. Although UCMS induced a reduced preference for the higher value reward in the judgement bias test, it did not affect saccharine preference or hypothalamic–pituitary–adrenal (HPA) activity. However, UCMS affected responses to ambiguous (intermediate) cues in the judgement bias test. While control mice showed a graded response to ambiguous cues, UCMS mice of both strains did not discriminate between ambiguous cues and tended to show shorter latencies to the ambiguous cues and the negative reference cue. UCMS also increased bar-mouthing in CD-1, but not in C57BL/6 mice. Furthermore, mice with higher levels of stereotypic behaviour made more optimistic choices in the judgement bias test. However, no such relationship was found for stereotypic bar-mouthing, highlighting the importance of investigating different types of stereotypic behaviour separately

Cohesin forms fountains at active enhancers in C. elegans

Abstract Transcriptional enhancers must find their target genes both efficiently and specifically. Chromatin conformation capture revealed the critical function of three-dimensional chromosome segmentation by topologically associated domains (TADs) to limit the search space of enhancers for promoters in mammals. In nematodes, although more than 30’000 sequences with characteristic enhancer chromatin features have been identified, the autosomal genome is not segmented by TADs, raising the question of the mechanism directing enhancer-promoter specificity. Using high-resolution HiC, we show that enhancer loci correlate with 3D hairpin-like structures extending 10-50 kb from the enhancers, hereafter designated as fountains. Fountains are specific to active enhancers, accumulate the major somatic cohesin and disappear when the latter is cleaved in vivo . Fountains accumulate topological constraints and are enriched for topoisomerases and the negatively-supercoiled DNA binder psoralen. Short-term topoisomerase depletion leads to small-scale structural changes at the fountain tip. Functionally, fountain disappearance correlates with enhancer-proximal gene activation, suggesting fountains play a similar role as TADs and direct enhancer-promoter interactions, in particular for genes expressed in neurons. We directly observe this cell-type specific upregulation for the skn-1/Nrf gene in a pair of head neurons. Phenotypically, cohesin cleavage has a major impact on nematode movement and foraging attitudes, demonstrating that changes in neuronal gene expression impact nervous system function, reminiscent of pathologies caused by cohesin mutations in humans. Together, this study highlights a clear link between 3D genome organization at enhancers by cohesin, transcriptional gene regulation and animal behavior

Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.F.L. is supported by a HFSPO post-doctoral fellowship. The Pelet and Peter laboratories are supported by grants of the Swiss National Science Foundation (SNF), and work in the Peter laboratory is funded by the European Research Council (ERC), the Swiss Initiative in Systems Biology SystemsX (RTD project YeastX) and the ETH Zurich. In addition grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-33503 and FEDER), 2014 SGR (Generalitat de Catalunya) and the Fundación Marcelino Botín (FB). F.P. is a recipient of an ICREA Acadèmia award (Generalitat de Catalunya)