Professor Achillefs Kapanidis

Abstract:

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures. © JoVE 2006-2014. All Rights Reserved.

Abstract:

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer. © 2014 The Author(s).