Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast

Robertson, J. Brian; Stowers, Chris C.; Boczko, Erik M.; Johnson, Carl Hirschie

doi:10.1073/pnas.0809482105

Cited by 56 publications

(70 citation statements)

References 37 publications

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“…As shown above, damage to or impairment of the respiratory cytochromes of the cell resulted in a shortened HOC phase of the YRO when DO is low (or dropping) and reduced the time before the culture returned to an LOC mode of energy metabolism when DO is high or rising. In addition, the introduction of chemicals (metabolites) such as ethanol and acetaldehyde has been shown previously to result in differing degrees of YRO phase resetting, depending upon the phase of the oscillation at which those substances were introduced (14). This phase resetting in response to the artificial introduction of metabolites prematurely switches the YRO from an LOC phase to an HOC phase characterized by an immediate drop in DO in the culture.…”

Section: Visible Light Induces the Ros Stress Response But Ros Does Notmentioning

confidence: 99%

“…The YRO was established with the CEN.PK113-7D strain of S. cerevisiae in continuous culture in a Bioflo 115 reactor as described previously (14). White light was applied to the culture vessel by placing one, two, or three 65-W compact CWF floodlights (Lithonia Lighting) around the vessel's water jacket (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, S. cerevisiae generally is assumed to be unresponsive to visible light and moreover is not thought to exhibit light-anticipatory behavior that might be mediated by an oscillatory timekeeping mechanism. However, under certain conditions of nutrient-limited growth, yeast cultures exhibit an ultradian respiratory oscillation (yeast respiratory oscillation, or YRO), that is characterized by 1-to 6-h rhythms of oxygen consumption, metabolite production, cell division, and gene expression (11)(12)(13)(14)(15)(16) which have been proposed to allow yeast to anticipate rhythmic occurrences of oxidative stress and mitigate its effect on DNA replication (13,17). Originally, this oscillation was studied in populations of synchronized cells using slow-dilution continuous culture in bioreactors containing high cell densities.…”

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confidence: 99%

“…An oscillating culture of CEN.PK113-7D with a stable period was initiated with 0.9 L/min air as described (14). The gas supply to the culture was switched from air to 100% N 2 at 0.9 L/min for 3 min at each phase point.…”

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Visible light alters yeast metabolic rhythms by inhibiting respiration

Robertson

Davis

Johnson

2013

Proc. Natl. Acad. Sci. U.S.A.

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Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.M any organisms are exposed to visible light in their environment. Full sunlight can deliver up to 10 quadrillion photons of visible light·cm −2 ·s −1 (i.e., 2,000 μEinsteins·m −2 ·s −1 ), and cloud cover reduces this exposure only by a factor of 10. Even though sunlight provides photosynthetic energy to plants and a medium for the vision of many animal species, its highintensity rays damage living cells when light-absorbing molecules cannot safely disperse the energy that photons bring. Although the destructive capacity of UV light is widely appreciated, photons of visible light can be deleterious, e.g., by destroying cytochromes and thus affecting cellular respiration (1) or by producing reactive oxygen species (ROS) that cause damage to DNA, membranes, and other cellular components (2). To cope with the damaging effects of light, organisms have evolved different strategies ranging from the expression of shielding pigments, such as melanin and carotenoids (3), to active mechanisms that sense light and respond quickly to mitigate/repair damage, such as iris constriction to protect the retina (4, 5), light-avoidance movements by chloroplasts and mitochondria (6, 7), and the induction/activation of DNA photolyase (8). A third strategy to minimize damage from light is to anticipate and prepare for its effects through the use of cellular timing mechanisms such as a circadian clock (9).The budding yeast Saccharomyces cerevisiae lacks the wellcharacterized photoreceptors of many other organisms [e.g., cryptochromes, phytochromes, and the photoreceptors whitecollar 1 (WC-1), and rhodopsins] that sense light intensity and spe...

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Section: Visible Light Induces the Ros Stress Response But Ros Does Notmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Visible light alters yeast metabolic rhythms by inhibiting respiration

Robertson

Davis

Johnson

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Biosynthetic genes peak during the HOC phase, whereas autophagy and vacuolar genes peak during the LOC. In a continuous YMC culture, a fraction of the culture divides synchronously during each YMC period (4,10,11), indicating a coupling between the YMC and the CDC. However, the mechanism of this coupling remains unclear.…”

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confidence: 99%

Metabolic cycling without cell division cycling in respiring yeast

Slavov

Macinskas

Caudy

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

104

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Despite rapid progress in characterizing the yeast metabolic cycle, its connection to the cell division cycle (CDC) has remained unclear. We discovered that a prototrophic batch culture of budding yeast, growing in a phosphate-limited ethanol medium, synchronizes spontaneously and goes through multiple metabolic cycles, whereas the fraction of cells in the G1/G0 phase of the CDC increases monotonically from 90 to 99%. This demonstrates that metabolic cycling does not require cell division cycling and that metabolic synchrony does not require carbon-source limitation. More than 3,000 genes, including most genes annotated to the CDC, were expressed periodically in our batch culture, albeit a mere 10% of the cells divided asynchronously; only a smaller subset of CDC genes correlated with cell division. These results suggest that the yeast metabolic cycle reflects a growth cycle during G1/G0 and explains our previous puzzling observation that genes annotated to the CDC increase in expression at slow growth.T wo kinds of periodic behavior have been characterized in slowly growing yeast cultures. The first, the classical cell division cycle (CDC), consists of four phases (G0/G1, S, G2, and M) that are easily distinguished by morphological criteria. When the growth rate of budding yeast is slowed by mutations or chemicals inhibiting growth, the duration of the G1/G0 phase increases relative to the durations of the S, G2, and M phases (1). Recently, we confirmed and quantified this CDC trend (Fig. 1A) in chemostat cultures whose steady-state growth rate was controlled by limiting natural nutrients (2, 3). The second kind of cycle, the yeast metabolic cycle (YMC), was first observed more than four decades ago (4) as periodic oscillations in the oxygen consumption of continuous, glucose-limited cultures growing in a chemostat. Like the CDC, the YMC can be divided phenomenologically into two phases: the low oxygen consumption phase (LOC), when the amount of oxygen in the medium is high because the cells consume little oxygen, and the high oxygen consumption phase (HOC), when the reverse holds (SI Appendix). We also reported previously (3) similar growth-rate changes in the relative durations of the phases of the YMC (Fig. 1B). As the growth rate increases, the relative duration of the LOC decreases whereas the relative duration of the HOC increases (Fig. 1B), similarly to the analogous changes in the CDC.These changes in the relative durations of the phases affect the composition of asynchronous cultures, because single cells from asynchronous cultures cycle metabolically (5, 6) and thus the fraction of cells in a particular phase is proportional to the duration of that phase relative to the entire cycle period. The increase in the relative duration of a phase results in the increase in the fraction of cells in that phase, and thus an increase in the population-average expression levels of genes peaking during that phase. Consider, for example, a ribosomal gene that peaks in expression during the HOC phase; at slow growt...

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Development of Luciferin Analogues Bearing an Amino Group and Their Application as BRET Donors

Takakura

Sasakura

Ueno

et al. 2010

Chemistry An Asian Journal

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We systematically synthesized bioluminogenic substrates bearing an amino group on benzothiazole, quinoline, naphthalene, and coumarin scaffolds. They emit bioluminescence in various colors: red, orange, yellow, and green. An amino-substituted coumarylluciferin derivative, coumarylaminoluciferin (CAL), showed the shortest bioluminescence wavelength among substrates reported so far. Further, the fluorescence of CAL did not exhibit solvatochromism, which suggests that its bioluminescence is not susceptible to environmental factors. We applied CAL as an energy-donor substrate for a bioluminescence resonance energy transfer (BRET) system with click beetle red luciferase (CBRluc), a mutant of firefly luciferase, as the energy-donor enzyme and yellow fluorescent protein (YFP) as the energy-acceptor fluorophore, and obtained a clearly bimodal bioluminescence spectrum. Stable bioluminescence that is not influenced by environmental factors is highly desirable for reliable measurements in biological assays.

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Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast

Cited by 56 publications

References 37 publications

Visible light alters yeast metabolic rhythms by inhibiting respiration

Visible light alters yeast metabolic rhythms by inhibiting respiration

Metabolic cycling without cell division cycling in respiring yeast

Development of Luciferin Analogues Bearing an Amino Group and Their Application as BRET Donors

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