Understanding Protein Mobility in Bacteria by Tracking Single Molecules

Abstract

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.

Guided nuclear exploration increases CTCF target search efficiency

“…Since the kinetics of a reaction or binding event depend on the diffusive properties and nuclear organization of the reactants (Bénichou et al, 2010;Rice, 1985), understanding the molecular interactions that control a nuclear protein's target search mechanism and its overall distribution is essential to understanding its function. Target search mechanisms have been extensively probed in prokaryotes (Hammar et al, 2012;Kapanidis et al, 2018), where current models (Bauer and Metzler, 2012) suggest that proteins find their target sites through facilitated diffusion (Slutsky and Mirny, 2004). In this family of models, proteins are suggested to move according to two principal modes: (1) sliding in 1D along the DNA strand; (2) occasionally disassociating from the strand, diffusing in 3D, only to bind at a proximal DNA site.…”

ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data

“…Fluorescence microscopy has become a crucial tool in studying bacterial cell biology (1)(2)(3)(4)(5)(6)(7)(8). It is minimally invasive and allows for the study of living bacteria in a controlled environment and to monitor the motion and sub-cellular topologies of any proteinaceous factor (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) or nucleic acids (25)(26)(27)(28).…”

Identification of multiple kinetic populations of DNA-binding proteins in live cells

“…Singlemolecule live-cell imaging commonly relies on fluorescent proteins that are genetically fused to the protein of interest ( Fig. 1A) (17)(18)(19)(20). Tracking the fluorescence signal of thousands of molecules, one molecule at a time, enables the building of physical models, from which physical parameters such as diffusion constants and detachment rates from DNA can be determined.…”