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

被引：36

作者：

Gabriel, Christian H. ^{[1
,2
,3
,4
,5
]}

del Olmo, Marta ^{[6
]}

Zehtabian, Amin ^{[7
]}

Jaeger, Marten ^{[8
]}

Reischl, Silke ^{[1
,2
,3
,4
,5
]}

van Dijk, Hannah ^{[1
,2
,3
,4
,5
]}

Ulbricht, Carolin ^{[9
,10
]}

Rakhymzhan, Asylkhan ^{[11
]}

Korte, Thomas ^{[12
]}

Koller, Barbara ^{[1
,2
,3
,4
,5
]}

Grudziecki, Astrid ^{[1
,2
,3
,4
,5
]}

Maier, Bert ^{[1
,2
,3
,4
,5
]}

Herrmann, Andreas ^{[12
]}

Niesner, Raluca ^{[11
,13
]}

Zemojtel, Tomasz ^{[8
]}

Ewers, Helge ^{[7
]}

Granada, Adrian E. ^{[14
]}

Herzel, Hanspeter ^{[6
]}

Kramer, Achim ^{[1
,2
,3
,4
,5
]}

机构：

[1] Charite Univ Med Berlin, Berlin, Germany

[2] Free Univ Berlin, Berlin, Germany

[3] Humboldt Univ, Berlin, Germany

[4] Berlin Inst Hlth, Lab Chronobiol, Berlin, Germany

[5] Berlin Inst Hlth BIH, Berlin, Germany

[6] Charite Univ Med Berlin, Inst Theoret Biol, Berlin, Germany

[7] Free Univ Berlin, Dept Biol Chem & Pharm, Inst Chem & Biochem, Berlin, Germany

[8] Charite Univ Med Berlin, Berlin Inst Hlth BIH, Core Genom Facil, Berlin, Germany

[9] Charite Univ Med Berlin, Immune Dynam Rheumatol & Clin Immunol, Berlin, Germany

[10] Deutsch Rheuma Forschungszentrum DRFZ, Immune Dynam, Berlin, Germany

[11] Deutsch Rheuma Forschungszentrum DRFZ, Biophys Analyt, Berlin, Germany

[12] Humboldt Univ, Dept Biol, Mol Biophys, Berlin, Germany

[13] Free Univ Berlin, Vet Med, Dynam & Funct Vivo Imaging, Berlin, Germany

[14] Charite Univ Med Berlin, Charite Comprehens Canc Ctr, Berlin, Germany

来源：

NATURE COMMUNICATIONS | 2021年 / 12卷 / 01期

关键词：

CORRELATION SPECTROSCOPY; NEGATIVE FEEDBACK; GENE-EXPRESSION; CRYPTOCHROME; REVEALS; PHOSPHORYLATION; IDENTIFICATION; HETEROGENEITY; PERIODICITY; COMPONENTS;

D O I：

10.1038/s41467-021-24086-9

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The 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 (similar to 5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

引用

页数：15

共 99 条

[1] Single Live Cell Monitoring of Protein Turnover Reveals Intercellular Variability and Cell-Cycle Dependence of Degradation Rates [J].