ABSTRACT
Tracking the inheritance patterns of proteins (TrIPP) is a live-cell imaging technique used for tracking maternal protein segregation patterns between mother and daughter cells during asymmetric divisions of budding yeast. We use the photo-convertible fluorescent protein Dendra2 fused to a protein of interest (POI). Irreversible conversion from green to red fluorescence allows for parallel monitoring of old and new proteins for several generations. Single-cell quantitative image analysis of time-lapse microscopy gives synthesis and decay rates, as well as segregation patterns of the POI. For complete details on the use and execution of this protocol, please refer to Auboiron et al. (2021).
Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Mitosis , Saccharomyces cerevisiae/cytologyABSTRACT
Inheritance of chromatin-bound proteins theoretically plays a role in the epigenetic transmission of cellular phenotypes. Protein segregation during cell division is however poorly understood. We now describe TrIPP (Tracking the Inheritance Patterns of Proteins): a live cell imaging method for tracking maternal proteins during asymmetric cell divisions of budding yeast. Our analysis of the partitioning pattern of a test set of 18 chromatin-associated proteins reveals that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3p, Fpr4p, and Tup1p, which are preferentially retained in the mother. Low abundance proteins also tend to be retained in the mother cell with the exception of Sir2p and the linker histone H1. Our analysis of chromatin protein behavior in single cells reveals potentially general trends such as coupled protein synthesis and decay and a correlation between protein half-lives and cell-cycle duration.
