INTRODUCTION

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FtsZ is a highly conserved protein that appears to be present in all prokaryotes, where it has an essential role in cell division (16). It assembles at midcell into a cytoskeletal structure designated the FtsZ ring (Z ring) that directs the process of septation (2, 3). It was suggested that the Z ring formed through the polymerization of FtsZ, and all subsequent work supports this hypothesis (15). One activity of this ring is to recruit other division proteins to the division site, where they participate in formation of the septum (16).

FtsZ has limited sequence identity to eukaryotic tubulins, suggesting that it is an ancestral homologue (19). The recent solution of the structures of FtsZ and tubulin support this conclusion, as the structures are remarkably similar (13, 22). In addition to these sequence and structural similarities there is functional similarity, as both tubulin and FtsZ are GTPases that can polymerize into higher-order structures. Like tubulin's GTPase, FtsZ's GTPase activity increases dramatically with the protein concentration (5, 14, 28, 29). Both have a low basal rate at protein concentrations too low to support polymerization and a significantly increased rate at concentrations above the critical concentration needed for assembly. Tubulin assembles into microtubules that are composed of protofilaments, head-to-tail polymers of the dimer (7). FtsZ also assembles into protofilaments (8, 19), although it is not known how these protofilaments are arranged in the Z ring.

FtsZ readily polymerizes in the presence of DEAE-dextran, a polycation that also promotes tubulin polymerization (8, 19). In the presence of DEAE-dextran, the FtsZ protofilaments tend to bundle or form sheets. Assembly of FtsZ into bundles of protofilaments at neutral pH was also observed by Bramhill and Thompson (4) but was later reported to be due to an inadvertent drop in the pH below 6 (9). More recently, assembly of FtsZ into bundles of protofilaments was observed in the presence of 7 to 25 mM Ca²⁺, a concentration 1,000-fold higher than the physiological concentration (30). Although these various conditions have been used to promote FtsZ assembly, FtsZ can readily assemble in their absence (20). Under such conditions, FtsZ assembles into a mixture of single protofilaments and polymers that contain two to three protofilaments. Importantly, under these more physiological conditions the FtsZ polymers, like microtubules, are dynamic, depolymerizing as the GTP is exhausted (20). Thus, GTP hydrolysis plays a role in regulating FtsZ polymer dynamics similar to its role in dictating microtubule dynamics.

In our previous study, we used electron microscopy and centrifugation to characterize FtsZ polymerization (20). Here we report that FtsZ polymerization can be monitored by 90° angle light scattering, a technique that we used to examine FtsZ assembly under a variety of conditions, including exposure to various divalent cations.

	MATERIALS AND METHODS

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Purification and polymerization of FtsZ. Escherichia coli FtsZ was purified as described earlier (20). However, in that description the ultracentrifugation step was inadvertently omitted. High-speed centrifugation of the 10,000 rpm supernatant was done at 38,000 rpm for 1 h in a Beckman 50.2 Ti rotor to remove the membrane fraction. The supernatant was subjected to 30% ammonium sulfate fractionation. All steps before and after this step in the purification procedure are the same as described previously (20, 21).

Light scattering assay of FtsZ polymerization. FtsZ polymerization was measured by 90° angle light scattering in a Hitachi fluorometer (model F-3010) with both the excitation and emission wavelengths set at 350 nm and a slit width of 1.5 nm. FtsZ was added to a final concentration of 500 µg/ml (12.5 µM) or as specified in the appropriate buffer to a fluorometer cuvette with a 1-cm path length. The cuvette was then placed in a cuvette chamber that was maintained at 30°C by a circulating water bath, and data were collected for 8 min to establish a baseline. Then the cuvette was removed, and 1 mM GTP or any other nucleotide was added to achieve a final reaction volume of 300 µl. The reaction mixture was gently stirred with a pipette tip, and the cuvette was returned to the cuvette chamber for data collection for a specified period of time. The reading at time zero is the first reading taken after the cuvette was returned to the chamber following nucleotide addition. The elapsed time for the nucleotide addition step was 20 to 30 s. The net change in light scattering following nucleotide addition was plotted as a function of time. Although data were collected every 2 s, for purposes of plotting we used only data obtained every 30 s or 1 min and in some instances 2 min.

FtsZ GTPase activity. FtsZ was incubated in reaction mixtures with a final volume of 50 µl at 30°C. The components of the reaction mixtures are described where appropriate in the text. The reaction was initiated by adding 1 mM [-³²P]GTP (250 to 400 cpm/pmol). At indicated times, 5-µl aliquots were withdrawn and assayed for the liberation of ³²P exactly as described previously (18, 20).

	RESULTS

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FtsZ polymers can be measured by 90° angle light scattering. We have recently shown that GTP-dependent polymerization of FtsZ can be assayed by electron microscopy and centrifugation (20). The FtsZ polymers that are formed are 7 to 20 nm wide, depending on whether they exist as a single protofilament or a bundle of two to three protofilaments. Actin filaments and microtubules are 6 and 25 nm wide, respectively, and their assembly can be assayed by electron microscopy or centrifugation as well as by a more convenient light scattering assay offering real-time kinetics (11, 12). We therefore explored whether FtsZ polymerization could be assayed by light scattering since development of such an assay would be useful to further characterize FtsZ polymerization.

View larger version (87K): [in this window] [in a new window]   FIG. 1.   Assay of GTP-dependent polymerization of FtsZ by 90° angle light scattering, centrifugation, and electron microscopy. (A) Polymerization of FtsZ was assayed by 90° angle light scattering in a fluorometer. FtsZ at a concentration of 500 µg/ml (12.5 µM) in 294 µl of polymerization buffer (50 mM MES-NaOH [pH 6.5], 10 mM MgCl2, 50 mM KCl) in a fluorometer cuvette was incubated at 30°C for 8 min to establish a baseline. The reaction was initiated with the addition of nucleotide to 1 mM as indicated, and light scattering was monitored. The net change in light scattering ( light scattering) following nucleotide addition was plotted against time. (B) For the assay of FtsZ polymerization by centrifugation, 1 mM GDP or GTP was added to FtsZ (200 µg/ml [5 µM]) in 100 µl of polymerization buffer. The reaction mixtures were centrifuged for 15 min, and the FtsZ pellets obtained were analyzed by SDS-PAGE and Coomassie blue staining. (C and D) For analysis of FtsZ polymerization by electron microscopy, 1 mM GDP (C) or GTP (D) was added to FtsZ (200 µg/ml) in polymerization buffer at 30°C and incubated for 10 min. Samples were then prepared for visualization by electron microscopy.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Light scattering is proportional to FtsZ concentration. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at different concentrations, as indicated, was incubated in polymerization buffer for 8 min at 30°C, and polymerization initiated by the addition of 1 mM GTP. (B) To obtain the critical concentration for FtsZ polymerization, the average values of the net change in light scattering ( light scattering) during the steady state were plotted against FtsZ concentration. The intercept on the x axis is the critical concentration.

The dynamic nature of FtsZ polymers can be monitored by 90° angle light scattering. To verify the above interpretation of the light scattering data, we manipulated reaction conditions to vary the length of time for which GTP would persist in the polymerization reaction. First, we varied the GTP concentration. Polymerization was initiated by adding GTP at different concentrations to reactions containing FtsZ at 500 µg/ml (12.5 µM) in polymerization buffer (Fig. 3A). The initial change in light scattering was not noticeably affected by GTP concentration, but it was apparent, that with increasing concentrations of GTP, the steady state persisted longer. With 0.1 mM GTP there was a decline in light scattering within 1 min, reaching zero in 3 min, whereas with 1 mM GTP the light scattering was constant for about 20 min before it started to decline, reaching zero in 35 min. This result demonstrates that the length of the steady state is proportional to the GTP concentration and is consistent with the interpretation of the above results for assays in which the FtsZ concentration was varied.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Assessment of the dynamic nature of FtsZ polymers by light scattering. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at a concentration of 500 µg/ml (12.5 µM) was incubated in polymerization buffer for 8 min at 30°C, and poly- merization was initiated by adding GTP to various final concentrations as indicated. (B) FtsZ at a concentration of 500 µg/ml (12.5 µM) was incubated for 8 min at 30°C in polymerization buffer containing different concentrations of KCl as indicated, and polymerization was initiated by adding 1 mM GTP. (C) FtsZ at a concentration of 500 µg/ml (12.5 µM) in polymerization buffer was incubated for 8 min at 30°C, and polymerization was initiated by adding 0.25 mM GTP. After depolymerization, additional rounds of FtsZ polymerization were induced by the addition of GTP as indicated.

FtsZ polymerizes over a broad pH range. Our previous studies on FtsZ polymerization (20) and the experiments described here were done mostly at pH 6.5. We did report previously that polymer formation as assayed by electron microscopy occurred at pH 7.2 but did not appear as abundant as that at pH 6.5 (20). We therefore examined the effect of pH on FtsZ polymerization more thoroughly by light scattering, centrifugation, and electron microscopy. As seen in Fig. 4A, GTP-dependent polymerization was examined at pH 6.5, 6.9, 7.5, and 7.9. At each pH an increase in light scattering occurred, indicating polymers were formed. However, the steady-state period was shorter at the higher pHs than at pH 6.5. Upon measuring the GTPase activity at the higher pHs, we found that in each case the rate was 25% higher than at pH 6.5 (data not shown). This difference contributes to the decreased length of the steady state at these higher pHs, as the GTP would be consumed earlier. Also at pH 6.5, the initial change in light scattering was consistently 20 to 30% greater than at other pH values.

View larger version (122K): [in this window] [in a new window]   FIG. 4.   Effect of pH on FtsZ polymer formation. For polymerization at pH 6.9, 50 mM PIPES-NaOH was used; for pH 7.5 and 7.9, 50 mM HEPES-NaOH was used. The MgCl2 and KCl concentrations in these buffers were 10 and 50 mM, respectively. The standard polymerization buffer was used for the assay at pH 6.5. (A) Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. FtsZ at a concentration of 500 µg/ml (12.5 µM) was incubated at 30°C for 8 min in buffers at different pH values, and polymerization was initiated by adding 1 mM GTP. (B to D) For electron microscopy analyses of FtsZ polymers formed at different pHs, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 µg/ml (5 µM) in buffers at pH 6.5 (B), pH 6.9 (C), and 7.5 (D). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy.

Effect of Mg²⁺ on polymer formation. FtsZ requires Mg²⁺ for GTP hydrolysis (6, 18, 26) but not for GTP binding (18, 26). In the absence of Mg²⁺, FtsZ forms polymers as seen by electron microscopy and as assayed by centrifugation (20). The concentration of Mg²⁺ that we used for polymerization was 10 mM, and therefore it was relevant to carefully examine the effects of the Mg²⁺ concentration on polymerization. At all concentrations of Mg²⁺ examined, as well as in the absence of Mg²⁺, a significant increase in light scattering was detected (Fig. 5A). The initial change in light scattering was directly correlated with the Mg²⁺ concentration, indicating that polymer mass decreased with decreasing Mg²⁺ concentration. The initial increases in light scattering with 0, 2.5, and 5 mM Mg²⁺ were 36, 46, and 76% of the increase observed with 10 mM Mg²⁺ (Table 1). Importantly, in the absence of Mg²⁺ the change in light scattering remained constant for as long as 45 min, suggesting that polymers formed but did not turn over. A similar observation was made when polymer formation was assayed by centrifugation (20).

View larger version (87K): [in this window] [in a new window]   FIG. 5.   Effect of Mg2+ on FtsZ polymerization and GTPase activity. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at a concentration of 500 µg/ml (12.5 µM) was incubated at 30°C for 8 min in polymerization buffer with different MgCl2 concentrations as indicated. Polymerization was initiated by adding 1 mM GTP. (B) The FtsZ GTPase reaction was initiated by adding 1 mM [-32P]GTP to a reaction mixture at 30°C containing FtsZ at a concentration of 500 µg/ml in polymerization buffer with different MgCl2 concentrations as indicated. At indicated times, samples were assayed for 32P formation from [-32P]GTP as described earlier (18). For electron microscopy analyses of FtsZ polymers formed with different concentrations of MgCl2, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 µg/ml (5 µM) in polymerization buffer containing 10 mM (C), 5 mM (D), 2.5 mM (E), and 0 mM MgCl2 (F). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy.

FtsZ polymerization does not require Ca²⁺. In our previous polymerization studies (20) and in all experiments described here, Ca²⁺ was not added to the buffers because it was considered not required for polymerization of FtsZ. However, Yu and Margolin (30) reported that a relatively high concentration of Ca²⁺ (>7 mM) was required for polymerization. Therefore, we examined the effect of Ca²⁺ on polymerization by light scattering and electron microscopy. To rule out the possibility that low levels of Ca²⁺ contaminating our buffers influenced polymerization, we examined the effect of EGTA on the polymerization reaction. With 1 mM EGTA, the kinetics and quantity of polymer formation were virtually identical to those for the control without EGTA (Fig. 6A). Essentially the same results were obtained with 5 mM EGTA, although the initial change in light scattering was slightly less (data not shown). The polymers formed in the presence of EGTA (Fig. 6B) appear similar to those for the control (Fig. 5C). Thus, Ca²⁺ is not required for polymerization of FtsZ.

View larger version (114K): [in this window] [in a new window]   FIG. 6.   Effect of Ca2+ on FtsZ polymerization and FtsZ's GTPase activity. (A and C) FtsZ at 500 µg/ml (12.5 µM) was incubated at 30°C for 8 min in polymerization buffer with or without 1 mM EGTA (A) or with 10 mM Ca2+ (C). Polymerization was initiated with the addition of 1 mM GTP, and light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (B and D) For electron microscopic analyses of FtsZ polymers, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 µg/ml (5 µM) in polymerization buffer containing 1 mM EGTA (B) or 10 mM CaCl2 (D). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy. (E) The FtsZ GTPase reaction was initiated by adding 1 mM [-32P]GTP to a reaction mixture at 30°C containing FtsZ at a concentration of 500 µg/ml in polymerization buffer with different concentrations of CaCl2 as indicated. At indicated times, samples were assayed for 32P formation as described earlier (18, 20).

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

In this study we have shown that 90° angle light scattering can be used to investigate FtsZ polymerization. We found that Mg²⁺ is not required for assembly but is required for the dynamic behavior of the polymers. We found that Ca²⁺ is not required for FtsZ polymerization and observed that at high concentrations, it induced bundling of protofilaments and reduced the dynamic aspect of FtsZ assembly. We also found that FtsZ assembly occurred throughout the pH range from 6.5 to 7.9.

Since light scattering had not been previously applied to FtsZ polymerization, we felt it necessary to analyze this in some detail. The theoretical analysis of light scattering by very long rod particles indicates that the amount of scattered light is dependent only on the total weight concentration of the rods provided that the length of rods is much greater than the wavelength of the incident light (11). That this is indeed the case for FtsZ polymers was confirmed by using the data obtained by light scattering to estimate the critical concentration. The value of 2.5 µM that was obtained is similar to that obtained previously by centrifugation (20).

The light scattering assay confirmed the dynamic nature of the FtsZ polymers. Under the conditions that we used assembly occurred quickly, reaching a steady state by the time we began measurements. However, we could readily observe the disappearance of polymers which occurred as the GTP was consumed. This correlation between disappearance of the polymers and exhaustion of GTP occurred under a variety of conditions. By altering the GTP, FtsZ, Mg²⁺, Ca²⁺, or KCl concentration we could manipulate the time at which GTP was consumed, and this always coincided with the time at which the polymers disappeared. In addition, conditions that inhibited GTPase activity but not assembly, e.g., the removal of Mg²⁺ or the addition of 10 mM Ca²⁺, inhibited or reduced the dynamic nature of FtsZ polymers.

Our study revealed that FtsZ polymers form in the absence of Mg²⁺, indicating that GTP hydrolysis is not required for assembly. Such polymers were very stable, however, indicating that GTP hydrolysis is required for the polymers to depolymerize. Furthermore, in examining the effect of the Mg²⁺ concentration on FtsZ assembly, we found that the steady state persisted longer at 10 mM Mg²⁺ than at lower concentrations of Mg²⁺. This difference could be explained by the effect of Mg²⁺ on the GTPase activity. The activity was enhanced at lower Mg²⁺ concentrations (1 to 5 mM), resulting in a more rapid consumption of GTP and the more rapid disappearance of the polymers. At lower Mg²⁺ concentrations the protofilaments were also shorter, indicating that disassembly was more rapid.

Under our experimental conditions, the basic unit of FtsZ assembly is a protofilament. At higher Mg²⁺ concentrations, there is a tendency for the protofilaments to align along the long axis to give polymers with two to three protofilaments. It is likely that at 10 mM Mg²⁺, where this bundling is more noticeable, the increased bundling results in a slower turnover of FtsZ polymers that also reduces the rate of GTP hydrolysis. Since this bundling appears to be induced by the elevated Mg²⁺ concentration, the physiological significance is unclear. This bundling is also affected by pH. Our study revealed that FtsZ polymerization occurs over a considerable pH range (6.5 to 7.9), although within this pH range assembly is most efficient at pH 6.5, similar to results with tubulin (23). However, the protofilaments also displayed increased bundling at acidic pH, where the GTP hydrolysis is reduced. Again, these results are consistent with the increased bundling reducing the rate of GTP hydrolysis. Lu et al. (14) also suggested that increased bundling was associated with decreased GTPase activity.

We also examined the role of Ca²⁺ since Yu and Margolin (30) reported that 7 to 25 mM Ca²⁺ induced dynamic assembly of FtsZ. In our initial studies we did not add Ca²⁺, assuming it was not required for assembly or the dynamic behavior of FtsZ polymers. The possibility of contaminating Ca²⁺ influencing the polymerization was ruled out by demonstrating that EGTA had no effect on assembly. Addition of Ca²⁺ in the millimolar range reduced the dynamic aspect of FtsZ assembly and inhibited the GTPase activity. Interestingly, millimolar concentrations of Ca²⁺ do not affect the critical concentration, as Yu and Margolin (30) estimated a critical concentration in the range of 2.5 to 3 µM, similar to the critical concentration that we found in the absence of Ca²⁺. The high concentration of Ca²⁺ notably increased the amount of light scattered. Inspection of the polymers formed in the presence of 10 mM Ca²⁺ revealed that Ca²⁺ dramatically increased bundling of protofilaments, leading to very large bundles. At these high concentrations Ca²⁺ must promote lateral associations between protofilaments, causing bundling much like DEAE-dextran-induced assembly of FtsZ (19). The bundling of FtsZ protofilaments by polycations is just one example of the bundling of charged biopolymers by polycations (27).

We suspect that the effects of Ca²⁺ are unlikely to be physiologically relevant because of the high concentration (>2.5 mM) required for Ca²⁺ to effect FtsZ polymerization compared to the estimated free Ca²⁺ concentration of 0.1 to 1.0 µM in vivo (10). Therefore, in vivo Ca²⁺ is unlikely to influence FtsZ assembly directly, although we cannot rule out some indirect role, for example, through an effect on another protein that regulates FtsZ assembly. The failure of Yu and Margolin (30) to observe polymerization in vitro with less than 7 mM Ca²⁺ is likely due to the assay that they employed; the fluorescence microscopic assay using FtsZ-GFP probably requires large structures that are relatively stable for visualization. As we have shown here, this is precisely the effects on FtsZ polymerization caused by 10 mM Ca²⁺.

The very dynamic nature of FtsZ polymers observed in vitro raises questions about how they are stabilized in vivo. For example, Z rings once formed exist for some time before they are used in division (1). Also, Z rings appear to persist in cell division mutants that are blocked after Z-ring assembly (1) or in cells treated with an inhibitor of septal peptidoglycan biosynthesis (24), suggesting that the polymers that make up the ring are stable under these conditions. As we have shown here, the assembly of protofilaments into bundles, however they are induced, decreases the dynamics of FtsZ polymers. Thus, any mechanism that causes association of protofilaments in vivo would contribute to their stability. In analogy with other polymerizing systems, FtsZ polymers likely have stabilizing factors such as cross-linking, nucleating, and capping factors.