A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies

Marinkovic, Zoran S.; Vulin, Clément; Acman, Mislav; Song, Xiaohu; Meglio, Jean-Marc Di; Lindner, Ariel B.; Hersen, Pascal

doi:10.7554/elife.47951

Cited by 28 publications

(18 citation statements)

References 62 publications

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“…The GR of a population of cells is a well established indicator for both genetic and environmental effects. Due to the magnification and limited field of view in microscopy, the largest population of cells which can be observed are small colonies and even those can suffer from nutrient limitation ( Marinkovic et al 2019 ). We implemented a population GR assay using a microfluidic chip to provide a continuous nutrient supply where S. cerevisiae or S. pombe cells grow in a single layer (adapted from ( Frey et al 2015 )).…”

Section: Resultsmentioning

confidence: 99%

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Schmidt

Cuny

Rudolf

2020

G3 Genes|Genomes|Genetics

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Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

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Section: Resultsmentioning

confidence: 99%

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Schmidt

Cuny

Rudolf

2020

G3 Genes|Genomes|Genetics

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“…As shown by our work, this is especially important for standard upright microscopy, where the volume of medium between the slide and coverslip is very small. For S. cerevisiae, conditions which do or may increase respiration and thus make cells more prone to hypoxia, are: growth in media with nonfermentable carbon sources such as ethanol, glycerol, galactose and others; growth to high density in glucose medium, where the glucose is mostly converted into ethanol during fermentation; growth in colonies, where in certain regions, a shift from fermentative to respiratory growth is observed (Marinkovic et al, 2019;Váchová, Čáp, & Palková, 2012). For other organisms, including other species of yeast, which are commonly imaged using upright microscopy, the issue may be even more important, because many of them are obligate aerobes, and thus respire continuously.…”

Section: Discussionmentioning

confidence: 99%

Modulation of green to red photoconversion of GFP during fluorescent microscopy by carbon source and oxygen availability

Bidiuk

Agaphonov

Alexandrov

2020

Yeast

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Studies have reported on the ability of green fluorescent proteins to photoconvert into a red fluorescent form under various conditions, such as the presence of oxidants, hypoxia, as well as under benign conditions using irradiation with a 405 nm laser. Here, we show that in Saccharomyces cerevisiae yeast green fluorescent protein (GFP) (S65T) fused to different cellular proteins can easily photoconvert into a red form when cells are grown in media with nonfermentable carbon sources. This photoconversion occurs during standard microscopy between glass slide and coverslip but is completely prevented by imaging on pads of solid medium or in a large volume of medium on an inverted microscope. The observed effect was due to rapid hypoxia of cells with respiratory metabolism in standard conditions for upright microscopy.Photoconversion could be prevented by antioxidant treatment, suggesting that it proceeds via the effects of reactive oxidative species emerging in response to oxygen deficiency. Our results show the need for caution during upright microscopy imaging in conditions where there is active respiration and demonstrate simple approaches to prevent unwanted GFP photoconversion. They also provide easy means of performing photoconversion experiments on existing GFP-bearing cell lines, at least in the case of yeast.

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“…The various gradients experienced by cells in colonies [41] create selective pressures not experienced by planktonic cells in liquid culture. Cells on agar and cells in liquid culture behave similarly just after inoculation [36].…”

Section: Chronological Aging In Yeast Coloniesmentioning

confidence: 99%

Diverse conditions support near-zero growth in yeast: Implications for the study of cell lifespan

Gulli¹,

Cook²,

Kroll³

et al. 2019

Microb Cell

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Baker's yeast has a finite lifespan and ages in two ways: a mother cell can only divide so many times (its replicative lifespan), and a non-dividing cell can only live so long (its chronological lifespan). Wild and laboratory yeast strains exhibit natural variation for each type of lifespan, and the genetic basis for this variation has been generalized to other eukaryotes, including metazoans. To date, yeast chronological lifespan has chiefly been studied in relation to the rate and mode of functional decline among non-dividing cells in nutrient-depleted batch culture. However, this culture method does not accurately capture two major classes of long-lived metazoan cells: cells that are terminally differentiated and metabolically active for periods that approximate animal lifespan (e.g. cardiac myocytes), and cells that are pluripotent and metabolically quiescent (e.g. stem cells). Here, we consider alternative ways of cultivating Saccharomyces cerevisiae so that these different metabolic states can be explored in non-dividing cells: (i) yeast cultured as giant colonies on semi-solid agar, (ii) yeast cultured in retentostats and provided sufficient nutrients to meet minimal energy requirements, and (iii) yeast encapsulated in a semisolid matrix and fed ad libitum in bioreactors. We review the physiology of yeast cultured under each of these conditions, and explore their potential to provide unique insights into determinants of chronological lifespan in the cells of higher eukaryotes.

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A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies

Cited by 28 publications

References 62 publications

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Modulation of green to red photoconversion of GFP during fluorescent microscopy by carbon source and oxygen availability

Diverse conditions support near-zero growth in yeast: Implications for the study of cell lifespan

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