Bacterial Chemoreceptor Imaging at High Spatiotemporal Resolution Using Photoconvertible Fluorescent Proteins
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.
KeywordsBacterial chemotaxis Superresolution microscopy Photoactivation localization microscopy (PALM) Single-particle tracking Receptor clustering Membrane protein mobility
We thank J.S. Parkinson and G. Pinas for strains, plasmids, and helpful discussions; A.S.N. Seshasayee for strain HS1; and H.C. Berg and K.A. Fahrner for the gift of anti-FliC antibody. This work was supported by NWO/FOM and the Paul G. Allen Family Foundation.
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