Abstract
Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σF, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.
Highlights
Zuzana et al (3 more authors) (Accepted: 2020) Single-molecule optical microscopy of protein dynamics and computational analysis of images to determine cell structure development in differentiating Bacillus subtilis
We find that the stoichiometry and diffusion of tracked SpoIIE is dependent on its interaction partners and morphological changes, suggesting its roles in sporulation are influenced by oligomeric composition and mobility
We modelled the frictional drag coefficient in the cell membrane of SpoIIE foci as that due to a cylinder whose height h matches the width of the phospholipid bilayer (~3nm) with a radius given by parameter a, using a generalized method established previously to characterize the lateral diffusion of transmembrane proteins [37,38]
Summary
Phase/epifluorescence microscopy of SpoIIE at different stages. Membrane labelling, FM4-. The resulting SpoIIAA displaces F from the F:SpoIIAB complex allowing RNA polymerase binding and transcription of forespore-specific genes (Fig. 1B)[10,11] This in turn, triggers activation of E in the mother cell and establishes alternate programmes of gene expression which determine different cell fates (Fig. 1A). It has been shown that there is selective proteolysis of SpoIIE in the mother cell through the action of the membrane bound ATP-dependent protease, FtsH[20] It is proposed selective oligomerization at the forespore pole, protects SpoIIE from proteolysis in this compartment and further increases the concentration difference between the cell compartments. We detect higher order mobile, oligomeric SpoIIE, towards the cell pole, at the stage of sporulation when F becomes selectively activated in the forespore, as previously proposed[20]
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