The cell cycle is a highly coordinated process governed by cyclin-bound cyclin-dependent kinases (CDKs). While the interaction between cyclin and CDK are well-documented, the dissociation constants (Kd) between specific cyclin-CDK pairs within living cells remain poorly understood. Fluorescence cross-correlation spectroscopy (FCCS) enables the quantification of the Kd, but challenges remain in selecting an optimal pair of fluorescent molecules for FCCS in a living cell. In this study, we demonstrate that mNeonGreen and phycocyanobilin-bound miRFP670 represent a suitable pair for FCCS in living cells from the viewpoint of high photostability and low bleed-through. This fluorescent protein pair enables us to measure the Kd values of the cyclin-dependent kinase Cdc2 and B-type cyclin Cdc13 in fission yeast cells. Moreover, we roughly estimated the Kd values for 36 cyclin-CDK complexes, formed by 9 distinct cyclins and 4 CDKs, in mammalian cells, including unconventional cyclin-CDK pairs. These measurements suggest potential versatility of cyclin-CDK binding in cell cycle progression, with implications for understanding cell cycle regulation in both fission yeast and higher eukaryotes.
See details in Toyama et al., J cell Sci., 2025
Aika Toyama, Yuhei Goto, Yuhei Yamauchi, Hironori Sugiyama, Yohei Kondo, Atsushi Mochizuki, Kazuhiro Aoki (2025) Quantification of Cyclin-CDK dissociation constants using FCCS with green and near-infrared fluorescent proteins., Journal of cell science
Published in 2025 Jul 31 (Electronic publication in July 31, 2025, midnight )
(Abstract) The cell cycle is a highly coordinated process governed by cyclin-bound cyclin-dependent kinases (CDKs). While the interaction between cyclin and CDK are well-documented, the dissociation constants (Kd) between specific cyclin-CDK pairs within living cells remain poorly understood. Fluorescence cross-correlation spectroscopy (FCCS) enables the quantification of the Kd, but challenges remain in selecting an optimal pair of fluorescent molecules for FCCS in a living cell. In this study, we demonstrate that mNeonGreen and phycocyanobilin-bound miRFP670 represent a suitable pair for FCCS in living cells from the viewpoint of high photostability and low bleed-through. This fluorescent protein pair enables us to measure the Kd values of the cyclin-dependent kinase Cdc2 and B-type cyclin Cdc13 in fission yeast cells. Moreover, we roughly estimated the Kd values for 36 cyclin-CDK complexes, formed by 9 distinct cyclins and 4 CDKs, in mammalian cells, including unconventional cyclin-CDK pairs. These measurements suggest potential versatility of cyclin-CDK binding in cell cycle progression, with implications for understanding cell cycle regulation in both fission yeast and higher eukaryotes.