Dynamics of bacterial cell division: Z ring condensation is essential for cytokinesis

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis.

associated proteins modulate FtsZ filaments, cell wall synthesis, and the overall process of cell division.
First, in order to understand which of the divisome proteins in B. subtilis associate with each other and work together, we characterized their dynamics using single-molecule imaging, as associated proteins should have similar motions. We expressed HaloTag fusions of each protein either as a sole copy or, in the case of SepF, at low levels from an ectopic site. Cells were sparsely labeled with JF549-HaloTag Ligand (7) and imaged with Total Internal Reflection Fluorescence Microscopy (TIRFM). Just as single molecules of FtsZ and FtsA are immobile (5), single molecules of the ZBPs were stationary (Fig 1B, Movie S2), consistent with their binding to stationary FtsZ subunits within treadmilling filaments. In contrast, the late proteins all moved directionally, with velocity distributions similar to Pbp2B (Fig 1C-D, Movie S3). The divisomeassociated cell wall synthesis enzymes are known to function together (8,9), and these data also show that the DivIB-DivIC-FtsL trimeric complex (10) remains persistently associated with these enzymes as they move around the division site. Thus, the divisome is composed of two distinct dynamic subcomplexes: 1) a directionally-moving group of periplasmic-facing membrane proteins that includes the cell wall synthesis enzymes, and 2) a group of cytoplasmic-facing proteins that bind to the stationary subunits within treadmilling FtsZ filaments.
Next, we investigated the function of the stationary subcomplex. We first examined FtsZ treadmilling, both to build a detailed characterization of its in vivo polymer dynamics and to provide a baseline for further investigation of how the ZBPs might affect its dynamics. In addition to visualizing FtsZ filament motion, we also measured FtsZ's subunit lifetimes: While filament motion reports only treadmilling velocity, subunit lifetime measurements reflect both the treadmilling velocity and filament length (11,12) (Fig 2A). Single-molecule imaging of FtsZ (Fig 2B, S1, Movie S4) gave a mean lifetime of 8.1 seconds (Fig 2C), corresponding to t½ = 5.6 s, similar to measurements of Z ring recovery after photobleaching (t½ = 8 ± 3 s) (13). Interestingly, FtsZ subunit lifetimes were higher at the division site than elsewhere (Fig 2D), possibly due to proteins such as Min and Noc that destabilize FtsZ filaments away from the division site (14). Confirming that our lifetime measurements report on FtsZ treadmilling, lifetimes increased with FtsZ(T111A), a mutant that treadmills more slowly, and decreased when treadmilling was accelerated by MciZ expression (5) (Fig 2E). We also characterized the dynamics of the rest of the stationary subcomplex and found that the lifetimes of FtsA and the ZBPs were similar to or slightly shorter than that of FtsZ ( Fig 2F).
Combining our lifetime measurements with treadmilling velocity gives insight into the in vivo properties of FtsZ filaments. First, we estimate that the average FtsZ filament is ~230 nm long, similar to the lengths of purified FtsZ filaments measured by electron microscopy (200 ± 75 nm) (15). Second, assuming that treadmilling filaments elongate with a diffusion-limited on-rate of 5 µM-1 s-1 (16,17), the concentration of free FtsZ monomer in cells can be estimated to be ~1.3 µM, similar to the critical concentration of FtsZ measured in vitro (1-1.5 µM) (15). This agreement between the critical concentration in vitro and in vivo suggests that the dynamics of FtsZ polymers in vivo are intrinsic to the polymer itself and unaffected by other factors. However, past work has suggested that FtsZ filament dynamics are modulated by other proteins in vivo (2).
To resolve this, we next investigated whether ZBPs affected FtsZ dynamics in vivo. ZBPs have been proposed to regulate FtsZ filament dynamics, stability, GTPase activity, and bundling, based both on in vitro biochemistry and in vivo genetic interactions (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). We measured FtsZ's treadmilling velocity and subunit lifetime in strains as we changed the levels of individual ZBPs by deletion or overexpression (Fig 3A, S2-4, Table S3). None of these ZBP perturbations had any effect on FtsZ's treadmilling velocity. Knockouts of ZapA or SepF also did not change FtsZ subunit lifetime, and overexpression had only subtle effects ( Fig S3, Table S4). However, ∆ezrA cells had increased lifetimes, consistent with previous FRAP measurements of FtsZ-GFP (13), and EzrA overexpression decreased lifetime in a dose-dependent manner (Fig 3B, S4). EzrA's effect of decreasing subunit lifetime without changing treadmilling speed suggest that it shortens FtsZ filaments, which we confirmed using Structured Illumination-TIRF (SIM-TIRF) ( Fig 3C, Movie S5); however, it does so without changing FtsZ's treadmilling dynamics, likely by binding to FtsZ monomers and sequestering them from the polymerizing pool (Supplementary Text 1). Thus, the ZBPs individually do not affect FtsZ treadmilling in vivo.
Next, we examined how the ZBPs in combination affect FtsZ dynamics. While none of the ZBPs are individually essential, ∆sepF and ∆zapA are each synthetically lethal with ∆ezrA (2). We created a ∆ZBPs strain that lacked all ZBPs by knocking out sepF and zapA and depleting ezrA using a xylose-inducible promoter. We depleted EzrA for 7 hours, at which point cells were filamented, indicating that division was blocked. We additionally repeated this for all other synthetically lethal combinations of ZBPs ( Fig S5). In all cases, FtsZ treadmilling velocity and subunit lifetime were unchanged from the control (Fig 3D, S6, Movie S6). We note that, although ∆ezrA cells have longer FtsZ subunit lifetimes, the lifetimes under these synthetic lethal conditions are statistically indistinguishable from the control. This suggests that the process that elongates FtsZ filaments in the absence of EzrA does not occur under these synthetic lethal conditions, i.e., in the absence of division and when cells are filamented (Supplementary Text 1). Regardless, together these data indicate that ZBPs do not affect FtsZ treadmilling in vivo.
However, in the absence of synthetically lethal combinations of ZBPs, cells showed severely altered Z rings: filaments no longer condensed, instead forming loose, regularly-spaced bands occupying regions ~1.6x as wide as control Z rings (Fig 3E-G, S5-6). These bands resemble the transient FtsZ structures that occur prior to Z ring condensation ( Fig 4A). Normally, these structures condense into Z rings prior to cell division (Fig 4B), but without ZBPs, condensation never occurs (Fig 3E,G). These results agree with previous observations that ZapA and SepF promote FtsZ bundling, whereas EzrA has previously been described as an inhibitor of Z ring formation and bundling (Supplementary Text 1). Here, we find that the ZBPs work collectively to promote Z ring condensation. Thus, without ZBPs, FtsZ filaments treadmill normally and localize correctly, but cannot condense into Z rings or divide the cell.
We next sought to clarify whether the Z ring condensation is specifically due to lateral bundling of FtsZ filaments by ZBPs. If this were the case, we might expect to isolate mutations that promote lateral bundling of FtsZ filaments in cells lacking ZBPs. Thus, we conducted a suppressor screen in the ∆ZBPs strain (see Supplemental Methods for details). Whole-genome sequencing of the resulting suppressor candidates revealed a charge-inverting mutation (K86E) in helix H3 of FtsZ; both this helix and the homologous residue have been shown to affect lateral FtsZ filament interactions in E. coli (29,30). We hypothesized that this mutation might restore viability in the absence of ZBPs by enhancing filament interactions. Indeed, FtsZ(K86E) restored viability and partially restored Z ring condensation in ∆ezrA ∆zapA cells (Fig 4C, S8).
Interestingly, the FtsZ(K86E) suppressor mutant can rescue the ∆ezrA ∆zapA cells but not other synthetic lethal combinations. Although the ZBPs work collectively to bundle FtsZ filaments, they may each affect bundling differently. Beyond their role as bundlers, the ZBPs have been shown to have distinct functions (2). Thus, the fact that FtsZ(K86E) can replace EzrA and ZapA but not SepF may reflect that each ZBP has different effects on FtsZ superstructure.
Finally, we investigated how the bundling of FtsZ filaments by ZBPs affected septal cell wall synthesis, which is required for cell division. Thus, we removed ZBPs and investigated the localization and motion of the division-specific cell wall synthesis enzyme Pbp2B, as well as septal cell wall synthesis activity (4D-E). Pbp2B recruitment to the Z ring decreased by 50% in ∆ZBPs relative to control cells ( Fig 4E); this is consistent with decreased Pbp2B recruitment at decondensed Z rings in control cells (Fig 4B). We found that in ∆ZBPs, Pbp2B nevertheless moved directionally at midcell; because the directional motion of Pbp2B reflects its activity, this suggests that it remains active under these conditions (Fig 4D, S9). To more directly assay the activity of cell wall synthesis enzymes, we measured the incorporation of fluorescent D-amino acids (FDAAs) into the division site (5); FDAA incorporation was still present in ∆ZBPs but reduced 40% relative to the control (Fig 4E, S9). Thus, septal cell wall synthesis enzymes are still active in the absence of the ZBPs, but Z ring condensation enhances their recruitment to FtsZ filaments at the division site.
Combined with past work, these experiments provide new insights into the mechanisms underlying bacterial cell division. The cell division process begins with short treadmilling FtsZ filaments that are restricted to midcell by negative regulators. Our data reveal that FtsZ filaments treadmill at their biochemical steady state; their dynamics are not modulated by other factors. However, FtsZ cannot form a functional Z ring on its own: ZBPs are also required to bundle FtsZ filaments into a condensed Z ring, transiently interacting with stationary FtsZ subunits without affecting filament dynamics. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, which move around the division site as part of a directionally-moving complex. This condensation is ultimately necessary for cell division (Fig 4F).
These results also yield new insights into the role of Z ring condensation in bacterial cytokinesis. Why is FtsZ filament bundling required for division, and what role does it play in the process? In contrast to previous models (29,31), FtsZ bundling does not modulate FtsZ treadmilling dynamics, but rather condenses the Z ring. While condensation is not required for the activity of division-associated cell wall synthesis enzymes, it may be necessary to concentrate their activity in a small enough region to allow for productive septation. It is also possible that lateral filament association serves to inwardly deform the membrane. FtsZ has been seen to deform liposomes when filaments coalesce (32,33), and crowding of membrane-associated proteins is sufficient to deform membranes (34). Such deformations may be easier if the periplasm is iso-osmotic with the cytoplasm (35,36), and therefore the force required for membrane deformation is small. This membrane deformation could promote late protein localization, or the late proteins might bind more readily to the early proteins once they have condensed; either mechanism could explain the enhancement of Pbp2B recruitment by Z ring condensation. This membrane deformation could then direct circumferential septal wall synthesis inward to divide the cell (5,37).

Fig. 2: FtsZ lifetime reports treadmilling dynamics in vivo.
A Left: Velocity and lifetime can be measured independently for a treadmilling filament. Velocity is measured by visualizing the motion of FtsZ filaments (dark gray circles), whereas lifetime is measured from the dwell time of a single labeled subunit (green circle) in the filament. Center: If the speed of a FtsZ filament is changed, this will result in a change in both the velocity and the lifetime that we measure. Right: If the length of a FtsZ filament is changed, the lifetime will change, but the velocity will not. Thus, lifetime reflects both treadmilling speed and filament length, whereas velocity is insensitive to filament length.  A The early Z rings before and after condensation in control cells. Each pair of images shows a newly formed Z ring that has not yet condensed (left), and the same Z ring after condensation (right). Z rings were visualized by epifluorescence imaging of FtsZ-mNeonGreen induced with 20 µM IPTG for 2 hours. Strain bAB219. B Left: Z ring condensation during the cell cycle. Top: Average intensity projections of Z rings over the cell cycle, created by averaging Z ring images from each normalized time point. Bottom: Z ring width over the cell cycle, measured as the full width at half maximum of the average intensity projections. Time from Z ring formation to Z ring disassembly (defined as the first and last frames in which the Z ring could