The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.

Synthesis of new septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo . How its activity is spatiotemporally regulated in vivo has also remained elusive. Here we confirmed FtsW as an essential septum-specific PGTase in vivo using an N -acetylmuramic acid analog incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

The bacterial tubulin FtsZ is the central component of the division machinery, coordinating an ensemble of proteins involved in septal cell-wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We used a combination of imaging, genetic and biochemical approaches to demonstrate that in Escherichia coli cells FtsZ exhibits dynamic treadmilling predominantly determined by its GTPase activity, and that the treadmilling dynamics directs processive movement of the septal cell-wall synthesis machinery. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall are substantially altered. Thus, the treadmilling of FtsZ provides a novel and robust mechanism for achieving uniform septal cell-wall synthesis to enable correct new pole morphology.One-sentence summaryThe bacterial tubulin FtsZ uses GTP hydrolysis to power treadmilling, driving processive synthesis of the septal cell wall.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.