Studying Cellular Dynamics Using Proximity-Dependent Biotinylation: Somatic Cell Reprogramming.

Abstract

Assessing the reorganization of proteins and organelles following the induction of reprogramming and differentiation programs is crucial to understand the mechanistic underpinning of morphological and fate changes associated with these processes. The advent of proximity-dependent biotinylation (PDB) methods has overcomesome ofthe limitations of biochemical purification methods, enabling proteomic characterization of most subcellular compartments. The first-generation PDB enzyme, the biotin ligase BirA* used in BioID, has now been used in multiple studies determining the cellular context in which proteins reside, typically under standard growth conditions and using long labeling (usually 8-24h) times. Capitalizing on the generation of more active PDB enzymes such as miniTurbo that can generate strong biotinylation signals in minutes rather than hours, as well as the development of an inducible lentiviral toolkit for BioID, we define here protocols for time-resolved PDB in primary cells. Here, we report the optimization and application of lentivirally delivered miniTurbo constructs to a mouse fibroblast model of somatic cell reprogramming, allowing the study of this dynamic process. This detailed protocol also provides a baseline reference for researchers who wish to adapt these techniques to other dynamic cellular processes.