Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells.
The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.
Department of Biochemistry, University of Oxford, Parks Road, Oxford, OX1 3QU, UK. stephan.uphoff@bioch.ox.ac.uk.
Stephan
S
Uphoff
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D000072760:Single Molecule Imaging / Q000379:methods*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins
DNA repair; DNA-binding proteins; Escherichia coli; Lambda red recombination; Single-molecule imaging; Single-particle tracking; Super-resolution fluorescence microscopy