Three-Dimensional Super-Resolution Imaging of the RNA Degradation Machinery in Caulobacter Crescentus

Abstract

Due the small size of bacteria and the diffraction limit (DL), obtaining precise information on the location of biomolecules through the use of conventional fluorescence imaging is difficult. However, the conventional resolution of optical microscopes (∼200 nm) can be circumvented by the time-sequential super-localization of single-molecule fluorescent labels, resulting in ∼5x resolution enhancement. We applied these super-resolution techniques, in combination with a double-helix point spread function microscope, to study the three-dimensional (3D) spatial organization of the RNA degradation machinery in Caulobacter crescentus (C. crescentus). The RNA degradosome plays a dominant role in mRNA degradation, a key step in gene expression. RNaseE is a key component in the RNA degradosome, and is thought to be the scaffold for this complex in C. crescentus. Previous diffraction-limited fluorescence imaging has shown that C. crescentus RNaseE exhibits a patchy localization throughout the nucleoid. We report the first 3D super-resolution images of RNaseE in live C. crescentus, with careful attention to remove any spurious clustering from over-counting of blinking fluorescent protein labels. We use the acquired localizations to quantify RNaseE's spatial distribution and find a non-random, clearly clustered distribution of this protein. Moreover, most RNaseE molecules are localized in regular clusters spaced along the cell axis, and it is possible to extract specific measures of cluster size and other parameters. Disturbing transcription perturbs the protein distribution, resulting in more diffuse RNaseE localization. Our results suggest that mRNA decay is spatially organized within C. crescentus, and that this organization is related to the function of RNaseE in mRNA homeostasis.