Cellular Distribution And Diffusivity Of Hfq With Interacting Rnas

In bacteria, Hfq plays key roles in small RNA (sRNA) mediated translational repression by either stabilizing the sRNA or mediating mRNA to sRNA interaction, leading translational repression and/or mRNA degradation by RNAse E (RNE) as one of the most general mechanisms. However, the cellular localization or distribution of Hfq remain controversial in the literature, and how such distribution is related to its interactions with mRNA or sRNA remain largely unexplored. To address these questions, we developed a rigorous immunofluorescence (IF) combined with fluorescence in situ hybridization (FISH), overcoming fixation or permeabilization artifacts. Using super-resolved IF and FISH images, we find RNE resides mostly on the membrane but Hfq and RNAs sRNA stays in the cytoplasm in the diffused manner. Then to further study the cellular dynamics of Hfq, we conducted single particle tracking photoactivated localization microscopy (sptPALM) on Hfq. By introducing point mutations to Hfq at different RNA binding faces, We discovered that Hfq diffusivity is dependent upon its interacting RNAs.