Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

AbstractThe cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

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Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

10.1101/2020.02.28.967752 ◽

2020 ◽

Author(s):

Christian H. Gabriel ◽

Marta del Olmo ◽

Amin Zehtabian ◽

Silke Reischl ◽

Hannah van Dijk ◽

...

Keyword(s):

Circadian Clock ◽

Cell Biology ◽

Fluorescent Proteins ◽

Single Cells ◽

Current Model ◽

Circadian Clocks ◽

Circadian Oscillator ◽

Clock Proteins ◽

Circadian Clock Proteins ◽

In Cells

ABSTRACTThe current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (∼5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator. Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.

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Cell-permeable Circadian Clock Proteins

10.21236/ada405529 ◽

2002 ◽

Author(s):

Carl H. Johnson

Keyword(s):

Circadian Clock ◽

Clock Proteins ◽

Circadian Clock Proteins

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Circadian Clock Proteins in Prokaryotes: Hidden Rhythms?

Frontiers in Microbiology ◽

10.3389/fmicb.2010.00130 ◽

2010 ◽

Vol 1 ◽

Cited By ~ 21

Author(s):

Maria Loza-Correa ◽

Laura Gomez-Valero ◽

Carmen Buchrieser

Keyword(s):

Circadian Clock ◽

Clock Proteins ◽

Circadian Clock Proteins

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Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation

Frontiers in Neuroscience ◽

10.3389/fnins.2021.647589 ◽

2021 ◽

Vol 15 ◽

Author(s):

Atsushige Ashimori ◽

Yasukazu Nakahata ◽

Toshiya Sato ◽

Yuichiro Fukamizu ◽

Takaaki Matsui ◽

...

Keyword(s):

Subcellular Localization ◽

Circadian Clock ◽

Posttranslational Modifications ◽

Cultured Cells ◽

Circadian Oscillation ◽

Clock Proteins ◽

Circadian Clock Proteins ◽

Circadian Physiology ◽

And Behavior ◽

Robust Systems

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

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Ubiquitin ligase TRAF 2 attenuates the transcriptional activity of the core clock protein BMAL 1 and affects the maximal Per1 mRNA level of the circadian clock in cells

FEBS Journal ◽

10.1111/febs.14595 ◽

2018 ◽

Vol 285 (16) ◽

pp. 2987-3001 ◽

Cited By ~ 4

Author(s):

Suping Chen ◽

Jing Yang ◽

Lu Yang ◽

Yang Zhang ◽

Liang Zhou ◽

...

Keyword(s):

Circadian Clock ◽

Ubiquitin Ligase ◽

Transcriptional Activity ◽

Mrna Level ◽

The Core ◽

Clock Protein ◽

In Cells

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2PT229 The regulation of cyanobacterial circadian clock proteins KaiB-KaiC interaction by ATP(The 50th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.52.s143_5 ◽

2012 ◽

Vol 52 (supplement) ◽

pp. S143

Author(s):

Risa Mutoh ◽

Atsuhito Nishimura ◽

So Yasui ◽

Kiyoshi Onai ◽

Masahiro Ishiura

Keyword(s):

Circadian Clock ◽

Annual Meeting ◽

Clock Proteins ◽

Biophysical Society ◽

Circadian Clock Proteins

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Relationship between HIF-1 and Circadian Clock Proteins in Obstructive Sleep Apnea Patients—Preliminary Study

Journal of Clinical Medicine ◽

10.3390/jcm9051599 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1599 ◽

Cited By ~ 4

Author(s):

Agata Gabryelska ◽

Marcin Sochal ◽

Szymon Turkiewicz ◽

Piotr Białasiewicz

Keyword(s):

Obstructive Sleep Apnea ◽

Sleep Apnea ◽

Circadian Clock ◽

Protein Level ◽

Hypoxia Inducible Factor ◽

Apnea Hypopnea Index ◽

Clock Proteins ◽

Obstructive Sleep ◽

Healthy Control ◽

Circadian Clock Proteins

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia and associated with the disruption of circadian rhythm. The study aimed to assess the relationship between hypoxia-inducible factor (HIF) subunits, circadian clock proteins, and polysomnography (PSG) variables, in healthy individuals and severe OSA patients. The study included 20 individuals, who underwent PSG and were divided into severe OSA group (n = 10; AHI ≥ 30) and healthy control (n = 10; AHI < 5) based on apnea-hypopnea index (AHI). All participants had their peripheral blood collected in the evening before and the morning after the PSG. HIF-1α, HIF-1β, BMAL1, CLOCK, CRY1, and PER1 protein concertation measurements were performed using ELISA. In a multivariate general linear model with the concentration of all circadian clock proteins as dependent variables, evening HIF-1α protein level was the only significant covariant (p = 0.025). Corrected models were significant for morning and evening PER1 (p = 0.008 and p = 0.006, respectively), evening (p = 0.043), and evening BMAL protein level (p = 0.046). In corrected models, evening HIF-1α protein level had an influence only on the evening PER1 protein level. Results suggest that OSA patients are at risk for developing circadian clock disruption. This process might be mediated by subunit α of HIF-1, as its increased protein level is associated with overexpression of circadian clock proteins.

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