Description
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.
Strain
L972 (h-), YG181 (h+ ade6-M210 leu1-32 z::Padh1-mScarlet-I-HA-mNeonGreen<<nat), HS445 (h- 2L::Padh1-mCherry2-HA-mNeonGreen<<nat), HS565 (h- 2L::Padh1-spmNeonGreen<<nat 3R::Padh1-miRFP670<<kan 1L::synPCB2.1<<bsd), HS494 (h- 1L::Padh1-SynPCB2.1<<bsd cdc2-miRFP670<<hyg cdc13-spmNeonGreen<<kan), YG2004 (h- 1L::Padh1-SynPCB2.1<<bsd 2L::Padh1-mEGFP-miRFP670<<nat), YG2005 (h- 1L::Padh1-SynPCB2.1<<bsd 2L::Padh1-mStayGold-miRFP670<<nat), YG2006 (h- 1L::Padh1-SynPCB2.1<<bsd 2L::Padh1-mStayGold(Sp codon optimized)-miRFP670<<nat), YG2007-1 (h- 1L::Padh1-SynPCB2.1<<bsd cdc13-miRFP670<<kan cdc2-mNeonGreen<<hyg)