Real-Time De Novo Deposition of Centromeric Histone-Associated Proteins Using the Auxin-Inducible Degradation System

Abstract

Measuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the auxin-inducible degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and reexpression in the same experimental setup. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and reactivation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.