Quantitative Imaging of <scp>B1</scp> Cyclin Expression Across the Cell Cycle Using Green Fluorescent Protein Tagging and Epifluorescence

Karuna, Arnica; Masia, Francesco; Chappell, Sally Claire; Errington, Rachel J.; Hartley, Andrew M.; Jones, D. Dafydd; Borri, Paola; Langbein, Wolfgang Werner

doi:10.1002/cyto.a.24038

Cited by 5 publications

(5 citation statements)

References 42 publications

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“…We estimate that in RPE-1 cells the concentration of Cyclin B1 increases from approximately 20 nM in S phase to approximately 150 nM in late G2 phase. Our measurements agree with previous studies [ 48 , 55 , 56 ], but other works reported higher concentrations [ 57 – 59 ]. The variability is likely explained by differences in the cell lines measured and in the methodology.…”

Section: Discussionsupporting

confidence: 93%

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

et al. 2022

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Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein–protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

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Section: Discussionsupporting

confidence: 93%

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

et al. 2022

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“…We estimate that in RPE cells the concentration of Cyclin B1 increases from ~20 nM in S phase to ~150 nM in late G2 phase. Our measurements agree with previous studies (Beck et al, 2011; Karuna et al, 2020; Thomas et al, 2005), although other works reported higher concentrations (Arooz et al, 2000; Frisa and Jacobberger, 2009; Xu and Chang, 2007). The variability is likely explained by differences in the cell lines measured and in the methodology.…”

Section: Discussionsupporting

confidence: 93%

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved Fluorescence Correlation Spectroscopy

Barbiero

Cirillo

Veerapathiran

et al. 2022

Preprint

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Measuring the dynamics with which the regulatory complexes of the cell cycle machinery assemble and disassemble is a crucial barrier to our understanding that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of protein-protein interaction kinetics in living cells.Here, we apply Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

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“…Autophagy markers p62 and LC3B showed a similar pattern with that of p-EGFR and p-Drp1 ( Figure 7 F ). Cyclin B1 is primarily distributed in the M phase and serves as the master regulator of the G 2 /M transition 46 . CDK1 interacts with 9 different cyclins including cyclin B1 that becomes inactivated at the end of the M phase 47 .…”

Section: Resultsmentioning

confidence: 99%

Cold Atmospheric Plasma Boosts Virus Multiplication via EGFR(Tyr1068) Phosphorylation-Mediated Control on Cell Mitophagy

Han¹,

Shen²,

Nan³

et al. 2022

Int. J. Biol. Sci.

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Objectives: Vaccination still remains as the most effective approach for preventing infectious diseases such as those caused by virus infection, with cell-based vaccine manufacturing being one flexible solution regarding the spectrum of infectious disorders it can prevent. Rapid cell-based virus propagation can enable high yield of vaccines against viral diseases that may offer critical values in the industry when handling emergent situations such as the ongoing viral disease pandemic. Methods: Through investigating the phenomenon and biological mechanism underlying redox-triggered cell survival towards enhanced viral particle production, this study explores novel strategies for improved yield of viral particles at a reduced cost to meet the increasing demand on cell-based vaccine manufacturing against viral diseases. Results: We found in this study that cold atmospheric plasma (CAP), composed of multiple reactive oxygen and nitrogen species including H 2 O 2 , could effectively enhance virus replication via triggering cell mitophagy that was dynamically modulated by the p-EGFR(Tyr1068)/p-Drp1(Ser616) axis using IBRV and MDBK as the virus and cell models, respectively; and removing H 2 O 2 can further enhance virus yield via releasing cells from excessive G 0 /G 1 cell cycle arrest. The observed efficacy of CAP was extended to other viruses such as CDV and CPV. Conclusion: This study provides experimental evidences supporting the use of CAP as a modulator of cell survival including mitophagy and mitochondria dynamics, and makes CAP an interesting and promising tool for enhancing the yield of viral vaccines if translated into the industry.

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Quantitative Imaging of B1 Cyclin Expression Across the Cell Cycle Using Green Fluorescent Protein Tagging and Epifluorescence

Cited by 5 publications

References 42 publications

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved Fluorescence Correlation Spectroscopy

Cold Atmospheric Plasma Boosts Virus Multiplication via EGFR(Tyr1068) Phosphorylation-Mediated Control on Cell Mitophagy

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