Stoichiometry and Mobility Switching of a Morphogenetic Protein in Live Differentiating Cells

Spore formation following asymmetric cell division in Bacillus subtilis offers a model system to study development, morphogenesis and signal transduction in more complex organisms. Extensive biochemical and genetic details of its sporulation factors are known, however, the molecular mechanisms by which asymmetry is generated remain unclear. A crucial membrane phosphatase, SpoIIE, couples gene regulation to morphology changes, but how it performs different functions dependent on cell stage is unknown. We addressed this puzzle using high-speed single-molecule fluorescence microscopy on live B. subtilis expressing genomically encoded SpoIIE fluorescent protein fusions during sporulation. Copy number analysis indicated a few tens of SpoIIE at sporulation onset increasing to 400-600 molecules per cell following asymmetric cell division with up to 30% greater proportion in the forespore, corresponding to a concentration enhancement in the smaller forespore sufficient for differential dephosphorylation of an anti-sigma factor antagonist and activation of the forespore specific transcription factor, {sigma}F. Step-wise photobleach analysis indicates that SpoIIE forms tetramers capable of reversible oligomerisation to form clusters correlated with stage-specific functions. Specifically, low mobility SpoIIE clusters which initially localize to the asymmetric septum are released as mobile SpoIIE clusters around the forespore when phosphatase activity is manifested. SpoIIE is subsequently recaptured at the septum in a SpoIIQ-dependent manner. After mother cell engulfment of the forespore, SpoIIE is released as a mix of higher mobility clusters and tetramers. Our findings suggest that additional information captured in the changing state of multimerization and mobility enable one protein to perform different roles at different cell stages.\n\nSignificance/impactCertain bacteria undergo sporulation involving cells dividing asymmetrically. A crucial protein SpoIIE facilitates this morphological asymmetry and directly links it to asymmetry in gene expression. Here, we used specialized light microscopy, capable of observing single molecules, plus biophysics, genetics and biochemical tools, to monitor SpoIIE in single living bacteria in real time, allowing us to count how many molecules are present in different cell regions, and how mobile they are. We find that SpoIIE clusters and moves depending on development stages, indicating that it has different roles depending on other binding proteins and their cellular locations. Our results suggest that changes in molecular stoichiometry and mobility may be used as switches in more complex cell processes.