Professor Achillefs Kapanidis

Abstract:

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Abstract:

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

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.