Abstract

Most single-molecule experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of a limited number of interacting components. However, to understand biological mechanisms as they occur in vivo while maintaining the extra information provided by single-molecule detection, there is a need for performing such experiments in cellular contexts, and in particular in living cells.

Towards this goal, we have been single-molecule fluorescence to perform localization-based super-resolution imaging (specifically photoactivated localization microscopy, PALM ) and to study the subcellular localization, diffusion properties, abundance and clustering of DNA -binding proteins in bacterial cells. We have also developed physical methods for delivering fluorescent biomolecules in bacteria and observing single-molecule fluorescence and single-molecule FRET in the bacterial cytoplasm.

I will discuss PALM studies of the subcellular localization and copy number of RNA polymerase in bacterial cells grown under different physiological conditions, both in fixed and living bacterial cells; using this method, we have identified the spatial distribution of an immobile fraction that represents mainly RNAP polymerases engaged in promoter and transcribed regions.

I will also how describe how PALM -based single-molecule diffusion tracks of polymerases in living bacteria (middle and bottom panels) allowed us to visualize DNA -damage and DNA -repair processes in real-time and at the single-molecule level for the first time.

Our approaches should be useful for studying many processes in gene replication, expression, and maintenance in various types of cells.