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

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 biochemic...

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Autores principales: Barbiero, Martina, Cirillo, Luca, Veerapathiran, Sapthaswaran, Coates, Catherine, Ruffilli, Camilla, Pines, Jonathon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9240681/
https://www.ncbi.nlm.nih.gov/pubmed/35765818
http://dx.doi.org/10.1098/rsob.220057
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author Barbiero, Martina
Cirillo, Luca
Veerapathiran, Sapthaswaran
Coates, Catherine
Ruffilli, Camilla
Pines, Jonathon
author_facet Barbiero, Martina
Cirillo, Luca
Veerapathiran, Sapthaswaran
Coates, Catherine
Ruffilli, Camilla
Pines, Jonathon
author_sort Barbiero, Martina
collection PubMed
description 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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spelling pubmed-92406812022-06-29 Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy Barbiero, Martina Cirillo, Luca Veerapathiran, Sapthaswaran Coates, Catherine Ruffilli, Camilla Pines, Jonathon Open Biol Research 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. The Royal Society 2022-06-29 /pmc/articles/PMC9240681/ /pubmed/35765818 http://dx.doi.org/10.1098/rsob.220057 Text en © 2022 The Authors. https://creativecommons.org/licenses/by/4.0/Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, provided the original author and source are credited.
spellingShingle Research
Barbiero, Martina
Cirillo, Luca
Veerapathiran, Sapthaswaran
Coates, Catherine
Ruffilli, Camilla
Pines, Jonathon
Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title_full Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title_fullStr Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title_full_unstemmed Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title_short Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
title_sort cell cycle-dependent binding between cyclin b1 and cdk1 revealed by time-resolved fluorescence correlation spectroscopy
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9240681/
https://www.ncbi.nlm.nih.gov/pubmed/35765818
http://dx.doi.org/10.1098/rsob.220057
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