Understanding Protein Mobility in Bacteria by Tracking Single Molecules

• Published in: Journal of molecular biology Vol. 430; no. 22; pp. 4443 - 4455
• Main Authors: Kapanidis, Achillefs N., Uphoff, Stephan, Stracy, Mathew
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 26.10.2018; Elsevier
• Subjects: anomalous diffusion; bacteria; Bacteria - metabolism; Bacterial Proteins - metabolism; cytoplasm; Cytoplasm - metabolism; fluorescence microscopy; fluorescent proteins; image analysis; macromolecular crowding; Microscopy, Fluorescence; Multiprotein Complexes - metabolism; protein diffusion; Protein Transport; Single Molecule Imaging - methods; single-molecule tracking; target search; single-molecule tracking; GFP; PSF; macromolecular crowding; PALM; protein diffusion; FRAP; anomalous diffusion; target search; MSD
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2018.05.002

Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm. [Display omitted]