Yeast lacks dedicated photoreceptors; however, blue light still causes pronounced oscillations of the transcription factor Msn2 into lớn & out of the nucleus. Here we show that this poorly understood phenomenon is initiated by a peroxisomal oxidase, which converts light inlớn a hydrogene peroxide (H2O2) signal that is sensed by the peroxiredoxin Tsa1 và transduced to lớn thioredoxin, to lớn counteract PKA-dependent Msn2 phosphorylation. Upon H2O2, the nuclear retention of PKA catalytic subunits, which contributes to delayed Msn2 nuclear concentration, is antagonized in a Tsa1-dependent manner. Conversely, peroxiredoxin hyperoxidation interrupts the H2O2 signal và drives Msn2 oscillations by superimposing on PKA feedbaông xã regulation. Our data identify a mechanism by which light could be sensed in all cells lacking dedicated photoreceptors. In particular, the use of H2O2 as a second messenger in signalling is comtháng khổng lồ Msn2 oscillations & khổng lồ light-induced entrainment of circadian rhythms and suggests conserved roles for peroxiredoxins in endogenous rhythms.

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Sunlight is a prerequisite for life by providing a source of heat and energy for primary production. Most organisms exposed lớn light therefore maintain an ability to adapt their activities khổng lồ its presence. For example, many organisms, from cyanobacteria lớn humans1, maintain autonomous cycles of activity that correspond to day and night even in complete darkness2. The ability khổng lồ respond lớn light is a fundamental feature of such circadian clocks3. In the yeast Saccharomyces cerevisiae, light alters ultradian metabolic rhythms4, which have been proposed lớn constitute models for circadian clocks5. In addition, the Zn-finger transcription factor Msn2 rhythmically shuttles into lớn and out of the nucleus in response to lớn illumination6, in spite of the laông xã of specialized photosensory proteins such as opsins, phytochromes and cryptochromes in this organism7. Nuclear localization of Msn2 is inhibited by cyclic AMP-controlled protein kinase A (PKA)8,9 and stimulated by the phosphatases PP1 và PP2A10,11. The Msn2 rhythm was proposed to lớn originate from oscillations in cAMPhường produced at intermediate light intensities12, based on the observation that Msn2 fails khổng lồ oscillate in mutants deficient in PKA feedbachồng regulation13 và on the recapitulation of oscillations in a mathematical mã sản phẩm of the PKA signalling pathway. A careful analysis of the localization behaviour in single cells indicated that Msn2 localization typically passes through three distinct và successive sầu states in response to illumination: cytoplasmic, nucleo-cytoplasmic oscillatory & more permanently nuclear14. Such gradually increased nuclear localization of Msn2 presumably reflects a gradual decrease in PKA-dependent phosphorylation và altered levels of PKA/phosphatase signalling intermediate(s). However, the of any such intermediates & the mechanism by which PKA senses light remains unknown.

Typical 2-Cys peroxiredoxins are enzymes that reduce hydrogene peroxide (H2O2) via two catalytic cysteines và thioredoxin acting as the hydroren donor15,16. During catalysis, a small proportion of the primary catalytic (peroxidatic) cysteine becomes hyperoxidized to the sulfinylated (Cys–SO2H) khung, which inactivates the enzyme17,18. Peroxiredoxins have received increased attention because of their functions in suppressing malignant tumours19, in keeping the genome free of mutations20 và as conserved executors of the ability of caloric restriction khổng lồ slow down the rate of ageing15,16,21,22,23,24. In addition, peroxiredoxin hyperoxidation was shown lớn sustain transcription-independent circadian rhythms in organisms from the three kingdoms of life1,25,26,27. On a similar note, oscillations in peroxiredoxin hyperoxidation were shown khổng lồ resonate with metabolic tidal rhythms in the marine crustacean Eurydice pulchra & peroxiredoxins have sầu also recently been suggested lớn modulate yeast ultradian metabolic rhythms5,28, indicating conserved functions in maintaining endogenous molecular clocks. The observation that light stimulates H2O2 production in cultured mouse, monkey và humans cells via photoreduction of flavin-containing oxidases such as peroxisomal acyl-coenzyme A (CoA) oxidase29 & the messenger function of H2O2 in zebrafish, which couples the light signal to lớn the circadian clock30, raise the interesting possibility that H2O2 might be a conserved second messenger by which light affects cellular physiology in general and endogenous clocks in particular.

Here we show that in S. cerevisiae a conserved peroxisomal oxidase converts the light impulse into a H2O2 signal that is sensed by the peroxiredoxin Tsa1 và then transduced khổng lồ thioredoxin lớn inhibit PKA activity. We propose that peroxiredoxin-mediated H2O2 signalling establishes rhythmic Msn2 nuclear accumulation by superimposing on PKA feedback regulation. In particular, we report that the Tsa1-mediated signal counteracts the nuclear retention of the two most highly expressed of the PKA catalytic subunits, thereby antagonizing a process that contributes lớn delayed Msn2 nuclear localization in response to H2O2.

Light is signalled khổng lồ Msn2 through H2O2

The transcription factor Msn2 rhythmically shuttles into and out of the nucleus in response khổng lồ light6. To study this phenomenon, we used an experimental thiết lập enabling both single-cell (Fig. 1a–c và Supplementary Movie 1) and population analyses (Fig. 1d) of Msn2 localization dynamics14. In short, cells were exposed lớn xanh visible light (λ=450–490 nm) & imaged in a perfusion chamber using epifluorescence microscopy as previously described14. At a light intensity corresponding to lớn that on a sunny day (P=115 μW), Msn2 accumulated into the nucleus và then baông chồng lớn the cytoplasm on average 4.3±3.6 times per hour (average±s.d., n=248) và, following an initial lag of ∼20 min, the proportion of cells exhibiting Msn2 nuclear localization14 stabilized at around 25% (Fig. 1d). Higher light intensities (P=200 μW) lead to a higher proportion of cells exhibiting nuclear localization (Supplementary Fig. 1a) and eventually resulted in more sustained nuclear localization14,31. In the absence of light, Msn2 did not accumulate into the nucleus6.

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(a) Frames from Supplementary Movie 1 showing that Msn2-GFP (green) rhythmically shuttles into lớn & out of the nucleus upon illumination of cells with xanh light (intensity 115 μW). Nup49-mCherry (red) was used to visualize the nucleus. Scale bar, 5 μm. (b) Single-cell trace representing nuclear localization upon illumination of a wild-type (wt) cell, quantified by comparing the signal intensity in the nucleus with the signal intensity in the cytosol (see Methods). The dashed line indicates the threshold ratio of nuclear/cytosolic Msn2 signal for nuclear localization in population analyses14. (c,f) Msn2 nucleocytoplasmic localization trajectories in individual wt cells upon illumination (115 μW, c) or upon the addition of 0.4 mM H2O2 (f). The colour scale indicates the degree of nuclear localization. (d,g) Fraction of wild-type, ccp1Δ & pox1Δ cells (d) or cells overexpressing (o/e) CCP1 or not (g) displaying nuclear localization of Msn2 following illumination at 115 μW. The number of cells assayed in each analysis are indicated in brackets. (e,h) Cytosolic H2O2, measured as average Cys199-dependent HyPerRed fluorescence, upon illumination of wt (e) and pox1Δ cells (h) at the indicated light intensities. Error bars indicate s.e.m. The number of HyPerRed/ HyPerRedC199S cells assayed in each analysis are indicated in brackets.

We hypothesized that H2O2 might serve as the light-induced second messenger, because light stimulates H2O2 production in mammalian cells29 & because yeast cells lacking the oxidant-responsive transcription factor Yap1 grow poorly in light4. To substantiate this, we used the cytosolic genetically encoded H2O2 sensor HyPerRed32, which we reasoned would be less sensitive sầu to bleaching upon illumination with xanh light than green fluorescent protein (GFP)-based H2O2 probes (for example, HyPer33 và roGFP2-Tsa2ΔCR34). Upon the start of illumination, the HyPerRed signal initially stayed constant but already after a couple of minutes decreased steadily at a rate dependent on light intensity (Supplementary Fig. 1b,c), indicating that probe bleaching interfered with its use in H2O2 determination. Nevertheless, in cells expressing an H2O2-insensitive probe (carrying a C199S substitution in the OxyR H2O2-sensing domain32), fluorescence decreased faster and/or to lower levels than in cells expressing the H2O2-sensitive probe (Supplementary Fig. 1b,c), indicating that upon illumination the levels of cytosolic H2O2 increased (Fig. 1e). The Cys199-dependent HyPerRed fluorescence increased as a function of light intensity (Fig. 1e), suggesting that so vày the levels of cytosolic H2O2. In support of H2O2 causing Msn2 nuclear redistribution upon illumination, an increased number of cells lacking the mitochondrial cytochrome c peroxidase Ccp1 exhibited nuclear Msn2 (Fig. 1d & Supplementary Fig. 1d,e); these cells are deficient in H2O2 scavenging. In addition, in wild-type cells, Msn2 rhythmically shuttled between the nucleus & the cytoplasm upon addition of intermediate levels of H2O2 (0.4 mM, Fig. 1f) và this level of exogenous H2O2 produced a comparable, albeit more rapid, increase in cytosolic H2O2 (C199-dependent HyPerRed fluorescence, Supplementary Fig. 1f,g). Higher levels of H2O2 elicited sustained localization of Msn2 to lớn the nucleus (Supplementary Fig. 1h–j). Conversely, in cells overproducing Ccp1 (∼3-fold, Supplementary Fig. 1k,l), which scavenge H2O2 at an increased rate, light-induced Msn2 nuclear localization was abolished (Fig. 1g), suggesting that cytosolic H2O2 is both necessary & sufficient for the light response of Msn2.

A peroxisomal oxidase converts light inlớn a H2O2 signal

Flavin prosthetic groups play unique roles in redox reactions by their ability to lớn participate in both one- and two-electron transfer processes35. In addition, they act as chromophores in dedicated light receptors of the ‘light-oxygen-voltage’, the ‘blue-light sensing using Flavin’ và the cryptochrome families36. In these proteins, photoexcitation of the flavin group initiates signal transduction through intramolecular conformational changes and/or altered protein–protein interactions36. However, upon excitation, flavins also become sensitive to lớn reduction by cellular reducing agents & can subsequently transfer electrons khổng lồ molecular oxygene, leading to the production of H2O2 (refs 35, 37, 38). Such reactions of flavin in peroxisomal fatty-acyl CoA oxidase were proposed to lớn be the cause of xanh light phototoxithành phố in mammalian cells through H2O2 production29. The yeast homologue of peroxisomal acyl-CoA oxidase is Pox1. Strikingly, Pox1 deficiency substantially decreased both cytosolic H2O2 (Fig. 1h) and Msn2 nuclear localization upon illumination (Fig. 1d and Supplementary Fig. 1m,n). However, both cytosolic H2O2 & Msn2 nuclear localization increased normally upon the addition of exogenous H2O2 to lớn cells lacking Pox1 (Supplementary Fig. 1o–r), indicating that peroxisomal acyl-CoA oxidase affects the response to light via H2O2. These data suggest that a peroxisomal acyl-CoA oxidase can signal light through H2O2 production in an organism lacking dedicated light receptors.

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A peroxiredoxin acts as a receptor of light-induced H2O2

The peroxiredoxin Tsa1 (see Fig. 2a) is required for the nuclear accumulation of Msn2 in response to H2O2, but not to lớn other stimuli, for example, NaCl that also trigger this response39. Based on our finding that light uses H2O2 as a messenger, we therefore asked whether light-induced Msn2 nuclear localization similarly depended on Tsa1. Indeed, we found that Msn2 remained cytoplasmic upon illuminating Tsa1-deficient cells, as evidenced by a representative sầu single-cell trace (Fig. 2b) and by the strongly decreased fraction of cells displaying Msn2 nuclear localization (Fig. 2c). This was true also at higher light intensities (P=200 μW, Supplementary Fig. 2a), further underscoring the crucial role of H2O2 in yeast light sensing. Moreover, we found that Tsa1 catalytic cycling was required for signal transduction, as Msn2 remained cytosolic in cells carrying serine substitutions of the Tsa1 catalytic cysteines (Fig. 2a,c,d and Supplementary Fig. 2b–d). It is known that a small fraction of Tsa1 becomes hyperoxidized to lớn the sulfinic acid size upon each turn of the peroxiredoxin catalytic cycle và such hyperoxidation inactivates enzyme peroxidatic cycling17,18 (see Fig. 2a). To further demo the importance of enzyme cycling, we therefore analysed Msn2 localization in cells expressing tsa1ΔYF. This mutant lacks the carboxy-terminal YF motif và is therefore resistant to hyperoxidation40,41 (Supplementary Fig. 2k). The fraction of tsa1ΔYF cells displaying Msn2 nuclear accumulation increased markedly (Fig. 2d & Supplementary Fig. 2e). However, this effect was completely negated by serine substitution of C171, which decreased Msn2 nuclear accumulation baông chồng to the low levels observed in the tsa1C171S single mutant (Fig. 2d & Supplementary Fig. 2f). These observations thus further point khổng lồ the importance of Tsa1 peroxidatic cycling for light signalling to Msn2 và the inhibitory effect of enzyme hyperoxidation on this response.