The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro

The bacterial tubulin FtsZ polymerizes to form a discontinuous ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL), and a C-terminal conserved peptide (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The assembly of FtsZ protofilaments into dynamic clusters is critical for cell division, but the interactions between protofilaments and regulatory mechanisms that mediate cluster assembly and dynamics are unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL (ΔCTL) shows a dramatically increased propensity to form long multifilament bundles compared with wild type (WT). ΔCTL also displays a reduced GTP hydrolysis rate compared with WT, but this altered activity does not account for bundle formation, as reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts ΔCTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of the FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between FtsZ-ring structure and function in bacterial cytokinesis.

The bacterial tubulin FtsZ polymerizes to form a discontinuous ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL), and a C-terminal conserved peptide (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The assembly of FtsZ protofilaments into dynamic clusters is critical for cell division, but the interactions between protofilaments and regulatory mechanisms that mediate cluster assembly and dynamics are unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL (⌬CTL) shows a dramatically increased propensity to form long multifilament bundles compared with wild type (WT). ⌬CTL also displays a reduced GTP hydrolysis rate compared with WT, but this altered activity does not account for bundle formation, as reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts ⌬CTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of the FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between FtsZ-ring structure and function in bacterial cytokinesis.
Canonical cytoskeletal proteins in animal cells polymerize to form structural elements that provide shape and mechanical integrity to the cell. In bacteria, however, the cell wall is the primary structural element, maintaining cell shape and preventing lysis. The role of bacterial cytoskeletal proteins that impact cell shape is in the spatial and temporal regulation of cell wall synthesis (1,2). Cytoskeletal polymer assembly, structure, and dynamics collectively regulate local shape changes by constraining and/or directing cell wall remodeling enzymes (2). During cell division, the cytoskeletal protein FtsZ polymerizes to form the cytokinetic ring or "Z-ring" at the incipient division site and recruits over two dozen proteins, including cell wall enzymes (3)(4)(5). The Z-ring comprises discontinuous clusters of circumferentially aligned FtsZ protofilaments that are highly dynamic (6 -9). How protofilaments are arranged within the resolution-limited clusters and how they interact with each other is largely unknown.
Recent studies in Escherichia coli and Bacillus subtilis demonstrated that FtsZ protofilament clusters in the Z-ring undergo treadmilling motion (8,9). FtsZ treadmilling, in turn, drives the circumferential movement of cell wall enzymes, thereby directing local cell wall synthesis and remodeling toward constriction. The treadmilling of FtsZ clusters is presumably driven by the polymerization and depolymerization of FtsZ protofilaments, because GTP hydrolysis mutants that have reduced turnover in vitro lead to slower movement of clusters in vivo (8,9). In addition to turnover of polymers, other aspects of the Z-ring such as conformational changes within FtsZ, lateral interactions between protofilaments, and interaction with FtsZ-binding proteins likely regulate cluster movement and thereby local cell wall remodeling (4,10,11). For example, FtsZ-binding proteins such as ZapA in E. coli and FzlA in Caulobacter crescentus that cause cross-linking or bundling of FtsZ protofilaments in vitro are important for efficient cytokinesis through unclear mechanisms (12)(13)(14).
FtsZ has a tubulin-like GTPase domain that polymerizes on binding GTP (Fig. 1A) (15)(16)(17). GTP hydrolysis is stimulated by polymerization and destabilizes the monomer-monomer interface, leading to depolymerization (17,18). Under physiologically relevant conditions and in the presence of GTP, FtsZ predominantly forms straight or gently curved single protofilaments and/or double protofilament bundles in vitro by transmission electron microscopy (19,20). In cells, FtsZ protofilaments are recruited to the membrane by membrane-anchoring proteins that bind FtsZ through the conserved peptide at its extreme C terminus (CTC 2 or C-terminal conserved peptide) (3,(21)(22)(23)(24). Between the GTPase domain and the CTC is an unstructured region called the C-terminal linker or CTL that varies widely in length (2-330 amino acids) and sequence across species (21). FtsZ from most bacteria with a peptidoglycan cell wall that have a CTC have a minimum CTL of 9 amino acids (21). Studies in E. coli and B. subtilis suggest length-, flexibility-, and/or disorder-dependent roles for the intrinsically disordered CTL in determining FtsZ assembly and function in vivo (25)(26)(27).
In our previous study characterizing CTL function in C. crescentus, we observed that C. crescentus FtsZ lacking its 172amino acid-long CTL (⌬CTL) forms highly bundled protofilaments in vitro (Fig. 1A) (28). This mutant of ftsZ when expressed in vivo leads to filamentation due to cytokinesis failure but also causes dominant lethal local cell wall defects leading to cell envelope bulges and rapid lysis. We were surprised to find that a variant of FtsZ with a 14-amino acid CTL (L14, Fig.  1A) caused filamentation but no bulging or lysis. Moreover, the L14 variant formed less bundled structures in vitro (28). We hypothesized that the aberrant assembly properties of ⌬CTL in vitro underlie the defects in the structure and function of the Z-ring formed by ⌬CTL in vivo (28).
In this study, we sought to fully characterize the contributions of the CTL to the polymerization properties of FtsZ in vitro. To this end, we compared polymers formed by wild-type FtsZ with ⌬CTL and L14. We also included in our in vitro characterization a chimeric FtsZ variant with the Hyphomonas neptunium CTL in place of the C. crescentus CTL (HnCTL) (Fig.  1A). HnCTL is inefficient but functional for cytokinesis; while the HnCTL gene can replace ftsZ at its genomic locus in C. crescentus, the resulting cells have slower doubling time and heterogeneous cell length (28). Our in vitro characterization of the CTL variants of FtsZ and their interactions with FtsZ-binding factors has revealed that the CTL is important for protofilament turnover and lateral interaction. ⌬CTL tends to form long extended bundles that require GTP binding but not hydrolysis for their formation. The membrane-anchoring protein FzlC can disrupt bundle formation in a CTC-dependent manner, but the CTC itself does not contribute to bundling. Moreover, the FtsZ curving protein FzlA can no longer robustly form helical bundles with ⌬CTL. Overall, our study provides a biochemical framework to connect the assembly properties of these CTL variants in vitro with their in vivo Z-ring structures, dynamics, and effects on the regulation of cell wall modeling.

FtsZ-CTL contributes to polymer dynamics
To better understand the contributions of the CTL to FtsZ polymerization, we performed a series of biochemical assays using purified CTL variants, FtsZ (WT), ⌬CTL, L14, and HnCTL, in vitro. Because polymerization and depolymerization are coupled to GTP binding and hydrolysis, we first determined the GTP hydrolysis rate for each variant using a malachite green assay to detect inorganic phosphate liberated over time. We observed a statistically significant reduction in the rate of GTP hydrolysis for each CTL variant as compared with WT ( Fig. 1B), with ⌬CTL exhibiting the slowest rate ( Fig. 1B and supplemental Fig. 1 To monitor polymerization over time for the FtsZ CTL variants, we used right angle light scattering. Light scattering has been used as a readout of FtsZ polymerization, as polymers scatter more light than monomers and the amount of light scatter increases with the size of polymers (19,20). It is important to keep in mind, however, that light scattering is also influenced by monomer size and structure and by formation of higher order filament assemblies such as bundles. Using this method for reactions containing 10 mM MgCl 2 and 2 mM GTP, we found that all variants readily polymerized upon addition of GTP with no detectable lag phase at our time resolution, and we did not observe any obvious differences in their initial rates of increase in scatter (Fig. 1C). However, whereas WT and HnCTL resulted in similar light scatter values at steady state, steady-state light scatter values for ⌬CTL and L14 were significantly lower (Fig.  1C). This trend was also true at the lower MgCl 2 concentration of 2.5 mM (Fig. 1D).
Curiously, when we determined the extent of polymerization of the FtsZ CTL variants using high-speed pelleting in the presence of 10 mM MgCl 2 , we were surprised to observe a distinct trend for steady-state polymer mass. Although each CTL variant showed a reduced percentage in the pellet (representing polymer fraction) compared with WT, ⌬CTL and L14 were enriched in the pellet compared with HnCTL ( Fig. 1, E and F). We reason that the longer CTLs of WT and HnCTL may significantly contribute to the light-scattering properties of their resulting polymers and that formation of higher order structures by one or more of the CTL variants may also influence light scattering or propensity to pellet, leading to different results in these two assays.
Finally, we used limiting concentrations of GTP to monitor the decrease in light scatter as filaments depolymerize following consumption of GTP. We found that ⌬CTL, with the slowest GTP hydrolysis rates at this condition ( Fig. 1B), took longer to return to baseline compared with WT and the other CTL variants, suggesting a correlation between the effect of the CTL on GTP hydrolysis rates and polymer dynamics, as we would predict (Fig. 1G).

CTL influences lateral interaction between protofilaments
We next assessed the filament structures formed by the four CTL variants using negative stain transmission electron microscopy (TEM). We observed polymers with similar protofilament widths for the four variants but with distinct appearance and abundance depending on the nature of the CTL. Similar to previous reports for C. crescentus FtsZ (12,20,29,30), at 8 M protein concentration with GTP and 2.5 mM MgCl 2 WT FtsZ formed gently curved single filaments and occasional straight double-filament bundles ( Fig. 2A). HnCTL and L14 predominantly formed sparse, short single filaments under the same conditions. Strikingly, as reported earlier (28), ⌬CTL predominantly forms long multifilament bundles at 8 M protein concentration and 2.5 mM MgCl 2 . We did not observe these long bundled structures for the other variants ( Fig. 2A).

FtsZ CTL regulates polymer structure and dynamics
Increasing the concentration of MgCl 2 has been reported to promote lateral interactions between FtsZ protofilaments. We therefore explored the effect of high MgCl 2 concentration on polymer superstructure for the four FtsZ variants. WT FtsZ frequently formed two-or three-filament bundles (7-15 nm wide) at 10 mM MgCl 2 (Fig. 2, B and C). In contrast, under the same conditions ⌬CTL bundles often consisted of more than four filaments (ϳ20 -200 nm in width) and appeared as very long, straight structures frequently extending longer than 1 m (Fig. 2, B and C). At 4 M protein and 2.5 mM MgCl 2 concentration, we did not observe bundles for ⌬CTL (Fig. 2D). Compared with WT protofilaments, L14 and HnCTL protofilaments were sparse on the grids and were predominantly gentlycurved single filaments at either MgCl 2 concentration (Fig. 2, B and D).
We further investigated whether polymer stability and/or slow GTP hydrolysis contributed to the increased bundling observed for ⌬CTL in vitro. To test this, we used GMPCPP, a slowly hydrolyzed analog of GTP, to form stable protofilaments at high and low concentrations of MgCl 2 . By TEM, at 2.5 mM MgCl 2 concentration, the polymer structures were not significantly different from GMPCPP compared with GTP ( Fig. 3A). At 10 mM MgCl 2 , we readily observed large multifilament bundles of ⌬CTL protofilaments with GMPCPP similar to those observed with GTP (Fig. 3B). Although we observed more protofilaments for all the FtsZ CTL variants, and more interaction between protofilaments for WT, we failed to find the long, wide multifilament bundles characteristic of ⌬CTL for the other variants. We conclude that the ability to form long multifilament bundles is specific to ⌬CTL.

FtsZ CTL regulates polymer structure and dynamics
The influence of CTL on lateral interaction was also evident from the differences in light scatter profiles of the CTL variants with GMPCPP ( Fig. 4). At 2.5 mM MgCl 2 , we did not observe large differences in the kinetics or extent of light scatter over time with GTP compared with GMPCPP for the CTL variants (Fig. 4, A and B). Only WT showed significantly higher scatter with GMPCPP compared with GTP (Fig. 4B). However, at 10 mM MgCl 2 with GMPCPP, we observed a two-step increase in

FtsZ CTL regulates polymer structure and dynamics
light scatter for WT and ⌬CTL (Fig. 4C). For WT we observed an initial rapid increase immediately following the addition of nucleotide (similar to light scatter with GTP) and then a second slower, much larger increase about 5 min later, likely corresponding to polymerization into protofilaments and formation of bundles, respectively. For ⌬CTL, the initial rapid increase was almost immediately followed by the second increase (after about 2 min). Moreover, the secondary increase in light scatter was much faster and larger for ⌬CTL compared with WT ( Fig.  4, C and D). HnCTL and L14 only undergo a rapid one-step increase in light scatter after addition of GMPCPP, not obviously different from GTP (Fig. 4, C and D) likely due to the absence of higher order assemblies of protofilaments under these conditions. We confirmed that the second increase in light scatter for WT and ⌬CTL with GMPCPP at 10 mM MgCl 2 was indeed due to increased lateral interaction by observing polymer structure over time (Fig. 5). WT incubated with GTP and 10 mM MgCl 2 forms predominantly short single filaments after 30 s and both single filaments and double-or triple-filament bundles after 15 min (Fig. 5A). In contrast, ⌬CTL incubated with GTP and 10 mM MgCl 2 forms few straight bundles that become longer and thicker with time (Fig. 5B). The difference in polymer structure for WT and ⌬CTL over time was more apparent with GMPCPP and 10 mM MgCl 2 (Fig. 5, C and D). Although WT largely formed single gently curved protofilaments after incubation with GMPCPP for 30 s, we observed more double-and triplefilament bundles after 5 min or a longer incubation (Fig. 5C).
We also observed a few long four-or five-filament bundles for WT after incubation with GMPCPP for 25 min. In contrast, we almost exclusively observe long four-or five-filament bundles for ⌬CTL after incubation with GMPCPP for 30 s (Fig. 5D). ⌬CTL multifilament bundles were more prominent, longer and thicker after a longer incubation with GMPCPP. Taking our observations together, we infer that the CTL is important for reducing lateral interaction between FtsZ protofilaments and that a minimal CTL of 14 amino acids is sufficient to perform this role.
Curiously, HnCTL does not form prominent double-or triple-filament bundles under any condition tested despite having WT-like light scatter with GTP. The decreased ability of HnCTL to form higher order protofilament assemblies is further suggested by the lower percentage of HnCTL in the polymer fraction as observed by high-speed pelleting (Fig. 1, E and F). A technical explanation for the sparse, unbundled filaments observed for HnCTL by TEM might be that HnCTL does not stick to the grids for TEM as well as WT FtsZ. However, with GMPCPP it is clear from light scattering that WT FtsZ forms higher order structures, although HnCTL does not. Thus, the sequence of the CTL seems to play a role in maintaining optimal interfilament interactions in a way that is altered for HnCTL.

High KCl and MgCl 2 concentrations favor bundle formation
Because we observe large bundles of ⌬CTL at a higher concentration of MgCl 2 (10 mM), we decided to further investigate

FtsZ CTL regulates polymer structure and dynamics
the effect of salt on lateral interactions between protofilaments at higher KCl concentrations. We performed GTP hydrolysis rate measurements, light scattering, and EM imaging of polymers formed by WT FtsZ or ⌬CTL at low (50 mM) or high (300 mM) KCl concentrations and low (2.5 mM) or high (10 mM) MgCl 2 concentrations. We did not observe significant differences in the GTP hydrolysis rates of WT at the different KCl and MgCl 2 concentrations (Fig. 6, A and B). However, unlike WT, we observed significant differences in GTP hydrolysis rates of ⌬CTL depending on KCl and MgCl 2 concentrations. At 50 mM KCl concentration, ⌬CTL displayed significantly reduced GTP hydrolysis rates compared with WT at both 2.5 and 10 mM MgCl 2 concentrations (Fig. 6, A and B). At 2.5 mM MgCl 2 concentration, we were surprised to see a significant increase in GTP hydrolysis rates of ⌬CTL at 300 mM KCl concentration compared with 50 mM KCl concentration (Fig. 6, A and B). In fact, ⌬CTL displayed the most similar (but still statistically significantly different) GTP hydrolysis rates compared with WT at 300 mM KCl and 2.5 mM MgCl 2 concentrations (Fig. 6B). In contrast, at 10 mM MgCl 2 concentration, there was a significant decrease in GTP hydrolysis rate of ⌬CTL at 300 mM KCl concentration compared with 50 mM KCl concentration (Fig. 6, A and B), suggesting that the reduction in GTP hydrolysis rate of ⌬CTL compared with WT is further enhanced at 300 mM KCl and 10 mM MgCl 2 . Thus, we conclude that KCl concentration affects GTP hydrolysis rates of FtsZ and ⌬CTL differently, and in a MgCl 2 concentrationdependent manner.
Although the differences in the effects of KCl concentration on GTP hydrolysis rates of ⌬CTL at low and high MgCl 2 concentrations appear counter-intuitive, our observations from light scattering and EM imaging helped provide a possible explanation. At 50 mM KCl concentration, ⌬CTL displayed significantly reduced light scatter compared with WT following addition of GTP, irrespective of MgCl 2 concentration (Fig. 6C). This reduction in light scatter was also observable at 300 mM KCl and 2.5 mM MgCl 2 concentrations (Fig. 6C). However, at 300 mM KCl and 10 mM MgCl 2 , we observed a large increase in light scatter for ⌬CTL compared with WT following addition of GTP (Fig. 6, C and D). This increase appeared comparable with the secondary increase observed for ⌬CTL following addition of GMPCPP at 50 mM KCl and 10 mM MgCl 2 concentrations (Fig. 4, C and D).
Next, we observed by EM the polymers formed by WT or ⌬CTL at the different KCl and MgCl 2 concentrations after incubation with GTP for 15 min. Although FtsZ forms predominantly gently-curved single filaments at 50 mM KCl and 2.5 mM MgCl 2 concentrations, we observed double-or triple-filament bundles of FtsZ more predominantly with higher KCl and/or MgCl 2 concentrations (Fig. 7, A and B, and supplemental Fig.  2A). This effect of KCl and MgCl 2 concentrations on lateral interaction between protofilaments was even more dramatic with ⌬CTL. Although ⌬CTL filaments were predominantly short, curved filaments at 50 mM KCl and 2.5 mM MgCl 2 concentrations, we observed long, straight filament bundles along with single gently-curved filaments at 300 mM KCl and 2.5 mM MgCl 2 concentrations (Fig. 7, C and D, and supplemental Fig.  2B). Overall, at 2.5 mM MgCl 2 concentration, there appeared to be more filaments on the grid at 300 mM KCl concentration compared with 50 mM KCl concentration for ⌬CTL (Fig. 7, C and D). Moreover, these filaments appeared longer at the higher KCl concentration (Fig. 7, C and D, and supplemental Fig. 2B). Taking this observation together with the results from GTP hydrolysis rate measurements, we conclude that Interestingly, despite the increased GTP hydrolysis rate of ⌬CTL at 300 mM KCl and 2.5 mM MgCl 2 , we still observed long, straight multifilament bundles that are distinct from the double-or triple-filament bundles observed for either ⌬CTL (on the same grid) or WT. Moreover, these bundles are comparable in thickness to the structures observed for ⌬CTL at 50 mM KCl and 10 mM MgCl 2 . Most strikingly, we observed large multifilament bundles that are much longer and thicker than those observed in the presence of GTP under all other conditions tested for ⌬CTL at 300 mM KCl and 10 mM MgCl 2 concentrations (Fig. 7, C and D, andsupplemental Fig. 2B), providing an explanation for the large increase in light scatter observed for ⌬CTL at this condition. We also observed a large number of single protofilaments, similar to those we typically observe for WT, coating the grid in the background under the high KCl, high MgCl 2 condition for ⌬CTL. This likely reflects increased polymerization of ⌬CTL at higher KCl concentration, but it may also reflect an increased propensity of polymers to interact with the EM grid.
In light of these observations, we propose that the decreased GTP hydrolysis rate observed for ⌬CTL at this condition (300 mM KCl and 10 mM MgCl 2 concentrations) is likely due to decreased turnover of monomers in the abundant, large protofilament bundles observed by EM. Alternatively, increasing KCl concentration could have a stabilizing effect on protofilament bundles by negatively affecting GTP hydrolysis rate at high MgCl 2 concentrations. The latter possibility is less likely because KCl appears to have a positive effect on ⌬CTL's GTP hydrolysis rate at low MgCl 2 concentration, and it has no observable effect on WT FtsZ's GTP hydrolysis rate irrespective of MgCl 2 concentration.

FtsZ CTL regulates polymer structure and dynamics High MgCl 2 concentration does not increase polymerization of ⌬CTL
We observed bundles of ⌬CTL with GTP or GMPCPP more readily at 10 mM MgCl 2 compared with 2.5 mM MgCl 2 . This effect of MgCl 2 on increasing lateral interaction has been documented before for full-length E. coli FtsZ, with more two-or three-filament bundles at higher MgCl 2 concentrations (19). We see the same effect for C. crescentus FtsZ (WT) (compare TEM shown in Figs. 2, B and D, or 3, A and B). However, we see fewer protofilaments for ⌬CTL by EM and lower signal by light scattering at 50 mM KCl and 2.5 mM MgCl 2 as compared with 10 mM MgCl 2 . We reasoned that the effects of increased divalent cation concentration might be the result of increased polymer formation (i.e. increase in total protofilament mass), increased bundling, or both. To differentiate between these possibilities, we used the polymerization-specific fluorescence reporter, a tryptophan mutant of FtsZ (FtsZ L72W ), developed by the Erickson lab (29). This mutant has a tryptophan residue engineered on the polymerizing interface of FtsZ, the environment of which changes on polymerization resulting in a polymerization-specific increase in fluorescence signal. Unlike light scat-tering, the increase in tryptophan fluorescence on polymerization is minimally affected by bundling and is a more reliable readout of number of the longitudinal interactions.
Using this assay, we first added 0.1 mM GMPCPP to reactions containing 4 M FtsZ (with 10% FtsZ L72W ) or ⌬CTL (with 10% ⌬CTL L72W ) and 50 mM KCl and then initiated formation of polymers with 2.5 mM MgCl 2 (Fig. 8A). We observed a polymerization-dependent increase in tryptophan fluorescence for both WT and ⌬CTL, as expected. Then we added MgCl 2 up to 10 mM and continued to measure the fluorescence intensity. Because the excitation and emission wavelengths for right angle light scatter and tryptophan fluorescence are similar (350 -350 nm compared with 295-344 nm respectively), we also included FtsZ alone or ⌬CTL alone (with no tryptophan mutation) as controls for a possible increase in intensity readout as a result of scatter. For WT, with or without the tryptophan mutant, we failed to observe a change in intensity when MgCl 2 concentration was increased, suggesting that increased MgCl 2 does not change the polymer mass at steady state under these conditions. For ⌬CTL, we did observe an increase in signal when we increased the MgCl 2 concentration. However, this signal

FtsZ CTL regulates polymer structure and dynamics
increase was observed for the ⌬CTL alone control, as well, indicating that it was the result of bundling-induced light scatter. Indeed, when we subtract the fluorescence signal observed for the ⌬CTL alone control from that of ⌬CTL with the tryptophan mutant, we observe no additional increase in signal on increasing MgCl 2 concentration (Fig. 8B). These observations argue against the possibility that the increased bundling seen by EM and increased signal observed by light scattering at 50 mM KCl and high MgCl 2 concentration are due to increased protofilament mass.
We also observed a drop in tryptophan fluorescence signal back to baseline after some time for WT, potentially due to the eventual depletion of GMPCPP through slow hydrolysis (Fig. 8,  A and B). In the same time frame, we did not observe a similar decrease for ⌬CTL likely because it has a slow nucleotide hydrolysis rate compared with WT.
To confirm that the increase in signal observed for ⌬CTL with GMPCPP following the increase in MgCl 2 concentration is due to increased light scatter, we measured the amount of light scattered (at 350 nm excitation and 350 nm emission wavelengths) by 4 M FtsZ or 4 M ⌬CTL and 50 mM KCl after addition of 0.2 mM GMPCPP and subsequent additions of MgCl 2 to 2.5 mM concentration and finally to 10 mM concentration. As predicted from our tryptophan fluorescence assay, we observed a large increase in light scatter for ⌬CTL immediately following the increase in MgCl 2 concentration to 10 mM (Fig. 8C). In comparison, we only observed a modest increase in light scatter for WT (Fig. 8C) similar to our prior observation with 0.2 mM GMPCPP at 10 mM MgCl 2 concentration (Fig. 4, C and D). Taken together, our results indicate that at 50 mM KCl, increasing MgCl 2 concentration promotes higher order assembly of ⌬CTL protofilaments without significantly increasing longitudinal interactions.

FtsZ and ⌬CTL can copolymerize in vitro
Because ⌬CTL has a dominant effect on cell shape and division in cells, we hypothesized that ⌬CTL and WT FtsZ co-polymerize in vivo and that ⌬CTL exerts its propensity for increased lateral interaction in the resulting copolymers. Because the polymerizing GTPase domain is intact in ⌬CTL, we expected that WT and ⌬CTL could still copolymerize. To test this formally, first, we estimated the minimum concentrations required for polymerization (i.e. critical concentration) of the tryptophan mutants FtsZ L72W and ⌬CTL L72W by determining the concentration below for which no increase in tryptophan fluorescence was observed on addition of GTP. We observed no significant difference in tryptophan fluorescence with or without GTP for protein concentrations less than 0.6 M for FtsZ L72W and 0.4 M for ⌬CTL L72W at 2.5 mM MgCl 2 and 50 mM KCl concentrations (data not shown). We used these concentrations as estimated critical concentrations in the following experiments. As expected, using only FtsZ L72W below its estimated critical concentration for polymerization, we observed no increase in fluorescence on addition of GTP at 2.5 mM MgCl 2 concentration (Fig. 9A). When we added WT FtsZ to this reaction mixture, pushing the total protein concentration above the critical concentration for FtsZ polymerization, we observed a significant GTP-dependent increase in fluorescence (Fig. 9A). We inferred from this observation that WT FtsZ can copolymerize with FtsZ L72W and pushes the overall monomer concentration above the critical concentration, thereby resulting in polymerization and an increase in tryptophan fluorescence. Importantly, we also observed this fluorescence increase when we added ⌬CTL to the reaction mixture instead of FtsZ, indicating that FtsZ L72W could copolymerize with ⌬CTL as well as WT. We observed the same effect when we used ⌬CTL L72W instead of FtsZ L72W further confirming that FtsZ and ⌬CTL can copolymerize (Fig. 9B). Because we used

FtsZ CTL regulates polymer structure and dynamics
limiting concentrations of GTP (50 M), we could also observe the decrease in fluorescence intensity due to depolymerization following depletion of GTP. The amount of time taken until depolymerization was exactly as estimated from the individual GTP hydrolysis rates, dominated by the predominant species of monomers (FtsZ or ⌬CTL), further adding confidence to our results. We did not observe any significant increase in fluorescence signal at 344 nm on addition of GTP for 4 M FtsZ or ⌬CTL at 2.5 mM MgCl 2 (Fig. 9C), suggesting that the signal observed in the presence of tryptophan mutants are indicative FtsZ CTL regulates polymer structure and dynamics of polymerization and not background scatter from polymerization of the proteins lacking the tryptophan.
On confirming that FtsZ and ⌬CTL can copolymerize, we compared the polymer structures formed by the copolymers at different ratios of FtsZ to ⌬CTL using negative stain TEM (supplemental Fig. 3). We compared the structures observed for mixtures of ⌬CTL and WT FtsZ with those formed at the same concentrations of WT FtsZ alone at 50 mM KCl and 10 mM MgCl 2 concentrations with GTP. We observed more polymer density on the EM grids with increasing FtsZ/⌬CTL ratios (supplemental Fig. 3A). We also observed more bundles for FtsZ/ ⌬CTL combinations compared with WT alone (compare supplemental Fig. 3, A and 1st panel of B). We saw prominent long ⌬CTL-like bundles at the lowest FtsZ/⌬CTL ratio (1:9). At higher ratios (1:3 or 1:1), the polymers were predominantly double-or triple-filament bundles similar to WT. However, the very tight packing of WT FtsZ on the grids made it difficult to distinguish possible differences in the length and abundance of bundles formed by FtsZ alone versus copolymers. The slowed polymer turnover of ⌬CTL could potentially lead to a higher ratio of FtsZ/⌬CTL in the polymer at steady state compared with initial conditions, further complicating the interpretation of these observations by TEM.
To more clearly visualize whether a 1:1 copolymer of FtsZ/ ⌬CTL exhibits different filament architecture compared with individual polymers of FtsZ or ⌬CTL, we compared GMPCPPstabilized protofilaments formed with a mixture of 2 M FtsZ and 2 M ⌬CTL to those formed by 2 M FtsZ or 2 M ⌬CTL alone (Fig. 9D). The use of GMPCPP-stabilized filaments allowed us to also visualize a 1:1 mixture of separately prepolymerized protofilaments of FtsZ and ⌬CTL to better identify structures unique to copolymers. We observed polymer structures characteristic of both FtsZ (predominantly gently curved single filaments or two-or three-filament bundles) and ⌬CTL (long multifilament bundles) on the grids with a mixture of pre-polymerized protofilaments (Fig. 9D, mixed (2 M each)). FtsZ/⌬CTL copolymers had a range of polymer structures from individual curved protofilaments to long doublefilament bundles to large multifilament bundles (Fig. 9, D, copolymers (4 M), and E). Uniquely, the FtsZ/⌬CTL copolymers also formed very long, evenly spaced double protofilament bundles that were not seen for mixed or individual polymers of FtsZ and/or ⌬CTL (Fig. 9, D and E, double arrow). These long double-filament bundles were conspicuous, as they often were observed in evenly spaced parallel arrays on the grid.
Overall, our results confirm that FtsZ and ⌬CTL can copolymerize and that FtsZ/⌬CTL copolymers can have distinct structural properties, most notably propensity to bundle and length of polymers, depending on the ratio of FtsZ/⌬CTL. Although it is not clear whether ⌬CTL's propensity to form multifilament bundles is relevant in vivo, these observations are in agreement with the increased time taken to elicit the ⌬CTL bulging and lysis phenotype in the presence of WT FtsZ compared with in its absence (28).
It is also interesting to note that we still see long thick ⌬CTL bundles on EM grids on dilution after polymerization with GMPCPP (compare ⌬CTL (4 M) to ⌬CTL (diluted 1:1) in Fig.  9D), whereas WT FtsZ double-or triple-filament bundles were less obvious on dilution after polymerization with GMPCPP (compare FtsZ (4 M) to FtsZ (diluted 1:1) in Fig. 9D). This further suggests that the increased ⌬CTL interaction is not an artifact of crowding on the EM grids but an ability to form stable lateral interactions that is lacking in WT FtsZ.

Increased lateral interaction between ⌬CTL protofilaments is CTC-independent
We previously reported that ⌬CTL requires its extreme C terminus (C-terminal conserved peptide or CTC) to cause cell wall defects (28). In vivo, ⌬CTLC (FtsZ lacking both the CTL and CTC, Fig. 1A) forms elongated structures in the cytoplasm instead of Z-rings (28). In contrast, ⌬CTC (FtsZ lacking only its CTC, Fig. 1A) forms broad Z-rings. To test whether the CTC contributes to the increased lateral interaction between ⌬CTL protofilaments in vitro, we compared protofilaments formed by ⌬CTC and ⌬CTLC to ⌬CTL bundles in vitro by TEM. At low MgCl 2 and 4 M protein, ⌬CTC formed WT-like protofilaments, whereas ⌬CTLC formed very few long protofilaments (Fig. 10A). At high MgCl 2 and 4 M protein, although the ⌬CTC protofilaments looked indistinguishable from WT protofilaments, ⌬CTLC formed large multifilament bundles similar to ⌬CTL (Figs. 10B and 11A). This suggests that the GTPase domain is sufficient for polymerization and bundle formation, similar to observations for the GTPase domain of B. subtilis FtsZ (31). These observations also rule out the possibility that the ⌬CTL bundles are the result of a difference in orientation of the CTC with respect to the polymerizing GTPase domain. We also observed similarly decreased apparent GTP hydrolysis rates for ⌬CTLC and ⌬CTC compared with ⌬CTL and WT, respectively, suggesting that the effects of lacking the CTC on GTP hydrolysis and polymerization are independent of the Dilution of GMPCPPstabilized polymers reduces crowding on EM grids and helps in better visualization of polymer structures. Scale bar, 100 nm. *, single protofilaments; arrow, two-or three-filament bundles; #, multifilament bundles; pair of double arrowheads, groups of two-or three-filament bundles that run parallel.

FtsZ CTL regulates polymer structure and dynamics
effects of lacking the CTL (Fig. 10, C and D). At 2.5 mM MgCl 2 , 50 mM KCl, and 4 M protein concentrations, FtsZ, ⌬CTL, ⌬CTC, and ⌬CTLC showed GTP hydrolysis rates of 3.18 Ϯ 0.16, 1.99 Ϯ 0.09, 2.94 Ϯ 0.19, and 1.50 Ϯ 0.06 GTP FtsZ Ϫ1 min Ϫ1 , respectively. We conclude that the absence of the CTL is the primary contributor to the increased lateral interaction

FtsZ CTL regulates polymer structure and dynamics
between protofilaments in ⌬CTL and that there is no observable effect of the CTC on lateral interaction in this context.

FzlC binding reduces lateral interaction between ⌬CTL protofilaments
FtsZ-binding partners such as FzlA and ZapA have been implicated in increased lateral interaction (bundling) of protofilaments (12,13,32,33). The increased bundling seen with ⌬CTL appears more pronounced than the previously characterized effects of binding partners of C. crescentus FtsZ at similar polymerization conditions. Consequently, we investigated the influence of binding partners of FtsZ on the increased bundles observed for ⌬CTL. Although ZapA has been implicated in increased bundling of FtsZ in E. coli, no such effect has been observed for C. crescentus ZapA (30). WT FtsZ protofilaments look indistinguishable with or without ZapA (supplemental Fig.  3C). We also failed to observe any difference in lateral interactions or structures of protofilaments formed by ⌬CTL in the presence or absence of ZapA (supplemental Fig. 3C).
Next, we characterized the effects of FzlC on protofilament structure (Fig. 11). FzlC was recently identified as a membraneanchoring protein for FtsZ in C. crescentus (12,24). In vivo, FzlC binds specifically to FtsZ's CTC and recruits it to the membrane prior to the arrival of FtsA (12,24). In vitro, His 6 -YFP-FzlC can recruit FtsZ-CFP to membranes of giant unilamellar vesicles in a CTC-dependent manner (24). At either low or high MgCl 2 concentrations, we did not observe any effect of His 6 -YFP-FzlC (2 M) on protofilament structure for WT FtsZ (4 M) (Fig. 11B,  FtsZ). Surprisingly, however, we found that the presence of His 6 -YFP-FzlC almost completely prevented bundling of ⌬CTL at high MgCl 2 concentrations (Fig. 11B, ⌬CTL). In fact, the presence of His 6 -YFP-FzlC restores WT-like protofilament structures for ⌬CTL. The presence of His 6 -YFP-FzlC did not affect the formation of multifilament bundles by ⌬CTLC at high MgCl 2 concentrations, suggesting that FzlC's ability to disrupt ⌬CTL bundle formation requires its binding to the CTC (Fig. 11B, ⌬CTLC). His 6 -YFP-FzlC did not have an observable effect on ⌬CTC, except for an overall reduction in the number of filaments on the grid (Fig. 11B, ⌬CTC). Thus, FzlC binding disrupts the increased lateral interaction in ⌬CTL in a CTC-dependent manner.

⌬CTL-induced bundling is dominant to FzlA-induced bundling
FtsZ protofilaments form helical bundles in the presence of FzlA in vitro (12). A recent characterization of FzlA from our laboratory confirmed that FzlA does not require the CTL or CTC to bind FtsZ. 3 We therefore assessed the effects of FzlA on protofilaments formed by CTL variants by TEM (Fig. 12, refer

FtsZ CTL regulates polymer structure and dynamics
to supplemental Figs. 4 and 5 for larger images). Our laboratory recently determined that, although WT FtsZ robustly forms helices with His 6 -tagged FzlA at pH 7.2 (12), it more robustly forms helices with untagged FzlA at pH 6.5. 3 We therefore first examined the structures formed by the CTL variants with untagged FzlA at lower pH. Under these conditions, we observed helical bundles only for WT FtsZ (Fig. 12, B and D, and supplemental Fig. 4, B and D). However, we did observe an apparent stabilizing effect of FzlA on ⌬CTL and L14 polymers (Fig. 12, A and D, and supplemental Fig. 4). At 2.5 mM MgCl 2 and 50 mM KCl concentrations, we did not observe many protofilaments of ⌬CTL or L14 in the absence of FzlA (Fig. 12A and supplemental Fig. 4A). However, in the presence of FzlA, we readily observed WT-like single filaments for both proteins ( Fig. 12B and supplemental Fig. 4B). Moreover, we also observed straight double-and triple-filament bundles for ⌬CTL with FzlA under these conditions ( Fig. 12B and supplemental Fig. 4B). It is unclear whether this is an effect on protofilament stability in solution or on binding EM grids. At 10 mM MgCl 2 concentration, we observed thick bundles for ⌬CTL that were unaffected by the presence of FzlA (Fig. 10, C and D, and supplemental Fig. 4, C and D). Despite the increased number of protofilaments observed for L14 in the presence of FzlA, we still failed to observe very large bundles like those seen for ⌬CTL either at low or high MgCl 2 concentrations (Fig. 12, B and D, and supplemental Fig. 4, B and D). The structures formed by HnCTL appeared similar in the presence or absence of untagged FzlA at pH 6.5 and either low or high magnesium concentrations (Fig. 12, A-D, and supplemental Fig. 4).
We also assessed the effects of untagged FzlA on the structures of polymers formed by CTL variants at the presumably more physiological pH of 7.2 ( Fig. 12E and supplemental Fig. 5). Again, we observed helical bundles only for WT FtsZ, and we observed a stabilizing effect of FzlA on ⌬CTL and L14 ( Fig. 12E and supplemental Fig. 5, A and B). This effect was specific to FzlA because glutathione S-transferase, which has structural homology to FzlA but does not bind FtsZ, did not affect polymer structure or density on grids under identical conditions (Fig. 12F).
To use a more physiological pH while simultaneously favoring FzlA-induced bundling, we decided to use 8 M His 6 -FzlA and 4 M FtsZ or CTL variants at 2.5 mM MgCl 2 concentration and pH 7.2. Under these conditions, all of the linker variants formed helical bundles similar to those observed for WT FtsZ (Fig. 12G and supplemental Fig. 5C). We observed fewer and shorter helical bundles for ⌬CTL/His 6 -FzlA compared with the other CTL variants or WT. Surprisingly, we also observed flat spiral or coiled structures for ⌬CTL/His 6 -FzlA (Fig. 12G, denoted by @, and supplemental Fig. 5C, inset). These structures resemble a highly-curved filament or filament bundle coiling onto itself. We hypothesize that these structures could result from increased self-interaction laterally between adjacent turns of helical double-or triple-filament bundles. Overall, our observations indicate that the CTL can affect FzlA-induced protofilament bundling and curvature. This is surprising because FzlA does not require the CTL for binding FtsZ 3 and suggests that the CTL may influence the FzlA-FtsZ interaction through its intrinsic effects on the FtsZ assembly.

Discussion
In this study, we demonstrate a role for the intrinsically disordered C-terminal linker region of C. crescentus FtsZ in regulating polymer structure and dynamics in vitro. FtsZ variants lacking the CTL entirely (⌬CTL, ⌬CTLC) exhibit increased propensity to bundle and reduced GTP hydrolysis rate and turnover compared with the other CTL variants or WT (Fig.  10). The exceptionally long and thick straight bundles of ⌬CTL protofilaments form independently of GTP hydrolysis rates, because we see long, thick bundles only for ⌬CTL even with the slowly hydrolyzed GTP analog GMPCPP (Figs. 2, B and C, and  3, B and C). Combining biochemistry, spectroscopy, and electron microscopy approaches, we confirmed that these bundles are not artifacts of observing a poorly polymerizing or depolymerizing FtsZ mutant under conditions that promote crowding (high MgCl 2 or protein concentration). We confirmed that WT FtsZ and ⌬CTL can copolymerize and observed that FtsZ/ ⌬CTL bundled copolymers appear much longer than FtsZ polymers (Fig. 9). We show that FzlC binding to the CTC domain can disrupt the ability of ⌬CTL to form bundles (Fig.  11). This observation suggests a role for the membrane-anchoring protein FzlC in regulating FtsZ polymer structure and dynamics in a CTC-dependent manner. Additionally, it also suggests that ⌬CTL has the ability to form gently curved single filaments given the right conditions and/or binding partners, but intrinsically it prefers to form bundles. Finally, we described a previously unidentified role for the CTL in facilitating FzlA to form helical bundles of FtsZ ( Fig. 12 and supplemental Figs. 4 and 5). Overall, our characterization of the CTL variants and their interaction with FtsZ-binding proteins under different polymerizing conditions provide novel insights into the contributions of the CTL to FtsZ polymerization dynamics.
Based on our observations, we propose the following roles for the disordered linker in regulating lateral and longitudinal interactions between FtsZ monomers in protofilaments (Fig. 13). Following addition of GTP, WT FtsZ undergoes fast polymerization (activation, nucleation, and elongation), reaches a steady state characterized by dynamic turnover, annealing and fragmentation and transient lateral interactions, and eventually depolymerizes following exhaustion of GTP. The CTL likely plays a role in maintaining optimal interactions between protofilaments laterally, acting as flexible, charged "repulsive brushes" extending orthogonally around the protofilaments. Such a mechanism of regulating lateral interaction between polymers has been described for the intrinsically disordered C-terminal region of neurofilaments (34). Additionally, the CTL could influence longitudinal interactions between monomers through a similar electrostatic repulsion mechanism and thereby influence monomer on and off rates and/or fragmentation and annealing. In the case of ⌬CTL, lateral interactions are more likely to form and less likely to break apart, with a strong propensity for the protofilaments to form long multifilament bundles. Moreover, ⌬CTL might lack the repulsive interactions between adjacent monomers in a protofilament and therefore affect depolymerization and/or fragmentation. Together, these differences in lateral and longitudinal

FtsZ CTL regulates polymer structure and dynamics
interactions would result in more stable polymers and slower turnover of monomers for ⌬CTL.
We consistently observed reduced lateral interaction for HnCTL (165 aa CTL, Figs. 1A, 2, 3, and 12) protofilaments compared with WT (172 aa CTL, Fig. 1A), by EM, pelleting, and light scatter assays. It is interesting to note that although C. crescentus CTL has a net charge of Ϫ9 and pI of 4.66, HnCTL has a net charge of Ϫ18 and pI of 4.2. The reduction in lateral interaction for HnCTL, which has a more negatively charged CTL than WT, further supports our model that electrostatic repulsion between protofilaments mediated by the CTL is important for optimal lateral interaction and bundle formation. A similar observation has been reported earlier for B. subtilis FtsZ CTL variants; replacing the CTL (50 aa) of BsFtsZ with the CTL from E. coli (50 aa) or a 50-aa region of the CTL from Agrobacterium tumefaciens causes reduction in lateral interaction both by EM and light scatter (25). Whether the CTL sequence-dependent difference in protofilament bundling is due to CTL-CTL interactions or CTL-GTPase domain interactions between adjacent protofilaments needs further investigation, for example, by engineering CTL mutants to alter net charge and/or disorder and determining their assembly properties. An alternative explanation for the differences between WT and HnCTL could be that the CTL is in fact not disordered entirely but is capable of assuming secondary conformations that regulate interprotofilament interactions. However, the CTL sequence across species is predicted to result in intrinsically disordered peptides making this possibility less likely.
Across species of bacteria, many members of the division machinery, such as FzlA and the Zap family of proteins, have been implicated in promoting interactions between FtsZ protofilaments in vitro and/or regulating the density of protofilaments in the Z-ring (12,13,32,33,35,36). Genetic manipulations of these proteins result in cell division defects, suggesting a role for regulating interfilament interactions in the cytokinetic function of FtsZ (13,30). 3 At least for the formation of helical bundles in vitro, untagged FzlA appears to require the CTL (Fig. 12). We have no evidence that FzlA interacts with the CTL. Thus, it is possible that the different intrinsic polymerization properties or lateral interactions of different CTL variants affect the curvature or polymer dynamics required for forming helical bundles upon FzlA binding. The His 6 -tagged variant of FzlA can form robust helices with all linker variants (Fig. 12G), which might be due to altered interactions with FtsZ. Interestingly, His 6 -FzlA also causes ⌬CTL to form unique spiral or coiled structures that have never been observed for Figure 13. CTL regulates lateral interactions between FtsZ protofilaments. Schematic describing polymerization dynamics of FtsZ and highlighting the proposed roles for the CTL in regulating longitudinal and lateral interactions. FtsZ monomers bind GTP, nucleate, and assemble into short protofilaments that can elongate further and/or anneal together. GTP-bound monomers within the protofilaments can undergo GTP hydrolysis leading to a conformational change that results in destabilization of longitudinal interactions. The CTL might play a role in regulating monomer-monomer interactions that contribute to this destabilization. This destabilization leads to fragmentation and/or depolymerization. At steady state, the forward reactions, nucleation, elongation, and annealing, balance out the reverse reactions, depolymerization and fragmentation, as long as GTP-bound FtsZ monomers are available. FtsZ protofilaments can also form lateral interactions that are most likely disrupted prior to fragmentation and/or depolymerization. We propose that the CTL plays a role in regulating this lateral interaction by functioning as a repulsive brush around FtsZ protofilaments. In the absence of the CTL (or both the CTL and the CTC), lateral interaction between protofilaments is stronger and/or less specific, thereby favoring the assembly of protofilaments into stable bundles. Furthermore, bundle formation could reduce protofilament turnover by stabilizing protofilaments against fragmentation/depolymerization.

FtsZ CTL regulates polymer structure and dynamics
WT FtsZ with FzlA again suggesting that His 6 -FzlA might have non-canonical interactions with FtsZ.
Many proteins that bind the CTC of FtsZ have been shown to affect Z-ring structure in vivo and/or affect FtsZ polymer structure in vitro (24,(37)(38)(39). It is unclear if and how these proteins regulate polymerization of FtsZ without interacting directly with the GTPase domain, because the CTC and the GTPase domains are linked by a long unstructured CTL. In the context of ⌬CTL, the CTC does not obviously contribute to the regulation of protofilament structure or lateral interaction in C. crescentus FtsZ. However, FzlC binding to the CTC disrupts ⌬CTL bundling. FzlC is conserved in ␣-proteobacteria (12). It acts as a membrane-anchoring protein for FtsZ in C. crescentus and may play a similar role in related species (24). Whether FzlC is relevant for regulating inter-protofilament spacing in WT FtsZ is unclear. It is curious, however, that whereas ZapA and FzlA, which bind the GTPase domain of FtsZ (13, 33), 3 do not affect bundling of ⌬CTL protofilaments, binding of FzlC to the CTC region disrupts bundling. It is possible that the binding of FzlC to the CTC of ⌬CTL disrupts inter-protofilament lateral interactions at the GTPase domain by restricting the orientation of protofilaments. Alternatively, FzlC bound to the CTC of ⌬CTL may perform a repulsive function to limit lateral interaction similar to the mechanism we propose for the CTL itself. Interestingly, the effect of FzlC on preventing ⌬CTL bundles occurs in the absence of membrane, indicating that this ability of FzlC, if relevant in vivo, is independent from its function as a membrane anchor for FtsZ. Further in vitro studies on the interaction between FzlC and FtsZ would be required to understand the mechanism underlying this observation and to determine whether it extends to other factors that bind the CTC, such as FtsA. Differences in the effects of membrane-anchoring proteins such as FzlC or FtsA on FtsZ polymerization could contribute to the roles of these proteins in cell division, in addition to their abilities to recruit FtsZ protofilaments to the membrane.
How transient or dynamic are the bundles observed for ⌬CTL? Does WT FtsZ also form similar bundles that dissociate more quickly and are difficult to capture for TEM? Our data from experiments using GMPCPP suggest that reducing turnover of FtsZ does not result in the same extent of bundling as observed for ⌬CTL. Although TEM provides very high spatial resolution of protofilaments and polymer structure, it has two major limitations. First, we cannot observe changes in structures over time. Second, differences in staining and affinity for the carbon-coated grids could bias the observer toward large bundles and/or well spaced polymers. To overcome these limitations and resolve FtsZ polymerization dynamics, we would require high-resolution time-lapse microscopy and/or spectroscopy techniques.
Our characterization of the CTL variants and FtsZ mutants provides biochemical tools for resolving the roles of longitudinal and lateral interactions in regulating protofilament structures in vitro and in vivo. Similar studies of polymerizing proteins such as actin and microtubules in eukaryotes have potentiated a relatively thorough understanding of the regulation and function of their assembly properties. Our recent understanding that the Z-ring is more than a passive scaffold for the recruitment of cell wall enzymes and the role for its dynamics in regulating cytokinesis emphasizes the need for understanding its polymerization properties in molecular detail. Additionally, although our observations strengthen the correlation between polymerization dynamics in vitro and Z-ring structure and function in vivo, whether this correlation is suggestive of causation or the result of a confounding effect of the CTL on unrelated processes requires further investigation.