Bacterial Chemoreceptor Imaging at High Spatiotemporal Resolution Using Photoconvertible Fluorescent Proteins.

Autor: Solari J; AMOLF Institute, Amsterdam, The Netherlands., Anquez F; Laboratoire de Physique des Lasers, Atomes et Molécules, UMR CNRS 8523, Université Lille 1, Villeneuve d'Ascq, France., Scherer KM; Laser Analytics Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK., Shimizu TS; AMOLF Institute, Amsterdam, The Netherlands. shimizu@amolf.nl.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2018; Vol. 1729, pp. 203-231.
DOI: 10.1007/978-1-4939-7577-8_18
Abstrakt: We describe two methods for high-resolution fluorescence imaging of the positioning and mobility of E. coli chemoreceptors fused to photoconvertible fluorescent proteins. Chemoreceptors such as Tar and Tsr are transmembrane proteins expressed at high levels (thousands of copies per cell). Together with their cognate cytosolic signaling proteins, they form clusters on the plasma membrane. Theoretical models imply that the size of these clusters is an important parameter for signaling, and recent PALM imaging has revealed a broad distribution of cluster sizes. We describe experimental setups and protocols for PALM imaging in fixed cells with ~10 nm spatial precision, which allows analysis of cluster-size distributions, and localized-photoactivation single-particle tracking (LPA-SPT) in live cells at ~10 ms temporal resolution, which allows for analysis of cluster mobility.
Databáze: MEDLINE