The Use of SNAP Labeling to Study Cell Cycle Oscillatory Proteins.

Abstract

Tightly controlled degradation of specific regulatory proteins is crucial for transitioning to the next cell cycle phase, ensuring precise DNA replication and an equal distribution of chromosomes to provide genomic stability and avoid tumorigenesis. To study mitotic control at the metaphase-to-anaphase transition, a histone H2-GFP-based reporter system was established, allowing simultaneous monitoring of the alignment of mitotic chromosomes and cyclin B proteolysis. To depict the proteolytic profile, a chimeric cyclin B-SNAP reporter molecule that can be labeled with a fluorochrome-carrying SNAP substrate was generated for measurement of the decline in fluorescence intensity via live-cell imaging. This reporter system can be adapted for other cell cycle oscillatory proteins.