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Originally published In Press as doi:10.1074/jbc.M312473200 on March 22, 2004

J. Biol. Chem., Vol. 279, Issue 25, 25959-25965, June 18, 2004
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Ruthenium Red-induced Bundling of Bacterial Cell Division Protein, FtsZ*

Manas Kumar Santra{ddagger}, Tushar K. Beuria{ddagger}, Abhijit Banerjee, and Dulal Panda§

From the School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India

Received for publication, November 14, 2003 , and in revised form, March 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 µM ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4–10 µM of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
FtsZ plays a key role during the cell division of bacteria and some eukaryotic organelles (1, 2). FtsZ self-assembles into a dynamic cytokinetic ring, known as a Z-ring, at the center of the cell within 1–5 min after cell division and remains attached to the leading edge of the invaginating septum for 15 min (3–5). The Z-ring recruits all other division proteins at the division site and organizes the cytokinesis (6, 7). The probable sequence of recruitment of the Z-ring and division proteins in vivo is known; however, the factors controlling the assembly and disassembly of the Z-ring are yet to be identified.

FtsZ has a limited sequence homology with tubulin, it shares a common GTP binding motif with tubulin, it assembles into dynamic protofilaments in a GTP-dependent manner, and it has significant structural similarities with tubulin, suggesting that FtsZ is a prokaryotic homolog of tubulin (8–15). Purified FtsZ monomers assemble into single-stranded protofilaments with little or no bundling of protofilaments in a polymerization reaction that is believed to be isodesmic in nature (16). Several factors are known to affect FtsZ assembly dynamics and bundling of protofilaments in vitro (17–22). For example, DEAE-dextran and polycation lipid monolayer are shown to induce protofilament association into sheets (17). Recently, divalent calcium (1–10 mM) and monosodium glutamate (0.1-1 M) were found to stabilize polymers and induce bundling of protofilaments (18, 19). In addition, a neutral macromolecule Ficoll (200 g/liter) was found to induce bundling of FtsZ protofilaments into ribbon-like structures (23). Further, a basic protein ZipA induces bundling of FtsZ polymers both in vitro and in Escherichia coli (24–26). The bundling of FtsZ protofilaments is thought to play an important role in the functioning of the cytokinetic Z-ring during bacterial division (27, 28).

The known inducers are required at least in millimolar concentrations to induce bundling of FtsZ protofilaments in vitro (18, 19, 23). In this study, we show that ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6·4H2O, can induce bundling of protofilaments at low micromolar concentrations. Ruthenium red is a water-soluble hexavalent polycation with several major uses in biological studies. In several proteins, ruthenium red competes for the calcium-binding site, and it is used as a probe for calcium (29, 30). It inhibits the actin-activated myosin Mg2+-ATPase in smooth muscle, perturbs the Ca2+ pump of the smooth muscle plasma membrane, and blocks the Ca2+-induced Ca2+ release from sarcoplasmic reticulum in both skeletal and cardiac muscle (31, 32). However, ruthenium red binds to tubulin at a site distinct from the high affinity calcium-binding site, and it is the only known ligand that binds to the {alpha}-tubulin subunit (33). Equimolecular concentration of ruthenium red has been shown to inhibit microtubule polymerization completely and to disassemble the preformed microtubules (34). Here, we found that stoichiometric concentrations of ruthenium red induced the bundling of FtsZ protofilaments in the presence of GTP, enhanced sedimentable polymers mass, prevented polymer disassembly, and reduced the GTPase activity of FtsZ. Further, ruthenium red and calcium were found to share their binding sites on FtsZ.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ruthenium red, guanosine 5'-triphosphate (GTP),1 1,4-piperazinediethanesulfonic acid (PIPES), isopropyl-{beta}-D-thiogalactopyranoside, sodium dodecyl sulfate (SDS), glycine, bovine serum albumin (BSA), EDTA, and Tris-HCl were purchased from Sigma. All other chemicals used were of analytical grade.

Purification of FtsZ—Recombinant Escherichia coli FtsZ was overexpressed and purified from E. coli BL21 strain (DE3) by using ionexchange chromatography (diethylaminoethyl cellulose DE-52) followed by a temperature-dependent one-cycle glutamate-induced polymerization and depolymerization method, as described recently (19, 35). FtsZ concentration was measured by the Bradford method (41) using BSA as a standard and adjusting the final concentration of FtsZ using a correction factor of 0.82 for the FtsZ/BSA ratio (15). The purified protein was frozen and stored at –80 °C.

Preparation of Ruthenium Red—Ruthenium red was dissolved in 25 mM PIPES buffer, pH 6.8, and centrifuged at 52,000 x g at 4 °C for 30 min. The supernatant was passed through a 0.2 µM Millipore filter and stored at –80 °C. The concentration of ruthenium red in the stock solution was calculated on the basis of a molar absorption coefficient of 68000 M–1 at 535 nm.

Light Scattering Assay—Briefly, FtsZ (7.3 µM) in buffer A (25 mM PIPES buffer, pH 6.8 containing 2 mM magnesium chloride) was incubated for 5 min in the absence and presence of different concentrations of ruthenium red on ice. After addition of 1 mM GTP, the reaction mixture was immediately transferred to 37 °C and the kinetics of FtsZ polymerization was studied by 90° light scattering at 450 nm using a JASCO 6500 fluorescence spectrophotometer. To investigate the effect of magnesium on ruthenium red induced FtsZ polymerization, FtsZ (7.3 µM) in 25 mM PIPES buffer (pH 6.8) was polymerized with and without 2 mM magnesium in the presence of 1 mM GTP and 10 µM ruthenium red. Similarly, we investigated whether GTP was essential for ruthenium red-induced FtsZ polymerization. FtsZ (7.3 µM) was incubated with 10 µM ruthenium red on ice for 5 min, and then the polymerization reaction was initiated by monitoring light scattering in the absence and presence of 1 mM GTP at 37 °C.

Measurement of FtsZ Polymer Mass by Sedimentation Assay—FtsZ (7.3 µM) was polymerized in buffer A containing 1 mM GTP in the absence and presence of different concentrations of ruthenium red for 15 min at 37 °C. The polymers were sedimented at 52,000 x g for 20 min at 30 °C. The supernatants were collected without disturbing the pellets. The FtsZ concentrations in the supernatants were measured by the Bradford method using BSA as a standard, with a correction factor of 0.82 for the FtsZ/BSA ratio (15). The amount of polymer formed was calculated by subtracting the supernatant FtsZ concentration from the total protein concentration. Using 280,000 x g for 15 min, Mukherjee and Lutkenhaus (13, 22) obtained 27 and ~50% of the total FtsZ in the pellets (12, 22). We found that 30 and 46% of the total FtsZ were pelleted as polymers using 52,000 x g in the presence of 2 and 10 mM magnesium, which were similar to the previously reported findings (12, 22). Therefore, we used 52,000 x g for all sedimentation assays. Stability of FtsZ Polymers in the Presence of Calcium and Ruthenium Red—FtsZ (30 µM) in 25 mM PIPES buffer was polymerized in the presence of 5 mM magnesium chloride, 10 mM calcium chloride, and 1 mM GTP for 15 min at 37 °C. The polymeric suspension of FtsZ was then diluted 30 times to reach a final concentration of 1 µM in warm PIPES buffer containing different concentrations of either ruthenium red (0–10 µM) or calcium (0–10 mM). The diluted polymeric suspensions were incubated for an additional 10 min at 37 °C. The polymers were sedimented at 52,000 x g for 20 min. The fraction of total FtsZ pelleted in the absence and presence of different concentrations of the cations was determined by using Bradford reagent, as described in the previous paragraph.

Visualization of FtsZ Polymers by Electron Microscopy—Samples were prepared for electron microscopy as described in our recent report with minor modification (19) and inspected by using a Philips CS 200 electron microscope. Briefly, FtsZ (7.3 µM) in buffer A was polymerized with 1 mM GTP in the absence and presence of different concentrations of ruthenium red at 37 °C for 10 min. 40 µl of FtsZ assembly solution was placed on a carbon-coated grid (300 mesh size). It was then blotted dry and subjected to negative staining by 1% uranyl acetate and immediately blotted dry again. All of the electron micrographs were taken at 50,000x magnification.

GTPase Activity Measurement of FtsZ—The production of inorganic phosphate during GTP hydrolysis was measured by a standard malachite green sodium molybdate assay (19, 35). FtsZ (7.3 µM) in buffer A was mixed to different concentrations (0–10 µM) of ruthenium red at 0 °C. Then, GTP (1 mM) was added to each sample, and the hydrolysis reaction was started by transferring the reaction mixtures to 37 °C. The GTP hydrolysis reaction was quenched at the desired time intervals by adding 10% (v/v) of 7 M perchloric acid, and the quenched reaction solutions were kept at 0 °C until all of the time points were collected. The quenched reaction mixtures were then kept at 25 °C for 10 min and centrifuged for 10 min to remove aggregated proteins. 15 µl of supernatants were incubated with 900 µl of filtered malachite green sodium molybdate solution at 25 °C for 30 min. The production of phosphate ions was determined by measuring the absorbance at 650 nm. A phosphate standard curve was prepared using sodium phosphate.

Binding of Ruthenium Red to FtsZ—FtsZ (7.3 µM) was incubated with 10 µM ruthenium red in 25 mM PIPES for 30 min on ice. FtsZ-ruthenium red complex was then separated from the free ruthenium red molecules by passing the solution through a Sephadex G-25 column (12 x 1 cm). The fraction containing the highest FtsZ-ruthenium red was dialyzed extensively against PIPES buffer at 4 °C. The concentration of bound ruthenium red was calculated using a molar absorption coefficient of 68,000 M–1 at 535 nm. The incorporation stoichiometry was calculated by dividing the bound ruthenium red concentration by FtsZ concentration. Further, the stoichiometry of incorporation of ruthenium red in the presence of 10 mM calcium was also determined. Briefly, FtsZ (7.3 µM) was first incubated with 10 mM calcium for 30 min on ice. Then, 10 µM ruthenium red was added in the reaction mixture and incubated for an additional 30 min.

To examine whether calcium could displace ruthenium red from the FtsZ-ruthenium red complex, FtsZ (7.3 µM) was first incubated with 10 µM ruthenium red for 30 min on ice. Then, calcium (10 mM) was added to the reaction mixture and incubated for an additional 30 min on ice. Partial purification of the FtsZ-ruthenium red complex was achieved by gel filtration. After gel filtration, FtsZ-ruthenium complex was dialyzed extensively for 24 h at 4 °C against PIPES buffer in the presence and absence of 10 mM calcium. The incorporation stoichiometry of ruthenium red per FtsZ was determined as described earlier.

Determination of the Stoichiometry of Copolymerization of Ruthenium Red in FtsZ Polymers—FtsZ (7.3 µM) was incubated with different concentrations (2–10 µM) of ruthenium red in the presence of 1 mM GTP and 2 mM magnesium at 37 °C for 15 min. FtsZ polymers were spun down (52,000 x g at 30 °C) to separate free ruthenium red molecules from the polymer-bound ruthenium red. The polymeric FtsZ was dissolved in PIPES buffer. The concentration of polymer-bound ruthenium red was estimated spectrophotometrically after subtracting the appropriate background (see below). The incorporation stoichiometry was calculated by dividing the bound ruthenium red concentration by FtsZ concentration. Ruthenium red is highly soluble in water. To get an idea of the nonspecific precipitation (background signal) of ruthenium red, BSA (7.3 µM) was incubated with different concentrations (2–10 µM) of ruthenium red in the presence of GTP and magnesium for 30 min at 37 °C, and then the reaction mixtures were centrifuged exactly the same way as described for FtsZ. In the absence of FtsZ, the amount of ruthenium red precipitated under identical experimental conditions was negligible (1%). We used the amount of ruthenium red precipitated at each concentration of added ruthenium red in the absence of FtsZ as a background to correct the experimental data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Ruthenium Red on FtsZ Polymerization—FtsZ (7.3 µM) was polymerized in the absence and presence of different concentrations (1–10 µM) of ruthenium red. Ruthenium red strongly increased the light-scattering signal of the FtsZ assembly in a concentration-dependent manner (Fig. 1). For example, 2 µM and 10 µM of ruthenium red increased the light-scattering signal by 1.5 and 5 times, respectively, compared with control (in the absence of ruthenium red). A closer analysis of the light-scattering traces revealed that the light-scattering signal increased biphasically in the presence of ruthenium red. The fast phase of polymerization was completed within the first 30–50 s of polymerization reaction, which was followed by a slow phase. In the slow phase of the polymerization reaction, the light-scattering signal increased several fold in the presence of ruthenium red, suggesting that ruthenium red induced the bundling of protofilaments of FtsZ.



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  		FIG. 1.
Effect of ruthenium red on FtsZ polymerization. FtsZ (7.3 µM) was polymerized in buffer A containing 1 mM GTP at 37 °C in the absence ({circ}) and presence of 1 µM (), 2 µM ({square}), 4 µM ({triangleup}), and 10 µM ({blacktriangleup}) ruthenium red.

 
The effect of ruthenium red on the polymerization of FtsZ was also investigated by a sedimentation assay (Fig. 2). Under the conditions used, 30% of the total FtsZ was sedimented in the absence of ruthenium red. It was reported previously that 27% of the total FtsZ was pelleted as polymers in the presence of 2.5 mM magnesium at 280,000 x g (22). Ruthenium red significantly increased the efficiency of pelleting FtsZ polymers. For example, 2 µM ruthenium red increased the fraction of total pelleted FtsZ by 50% compared with control (in the absence of ruthenium red). At a relatively higher ratio of FtsZ: ruthenium red (1:1.4), the amount of pelleted polymer was increased by a factor of 2.



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  		FIG. 2.
Ruthenium red increased FtsZ polymer mass. FtsZ (7.3 µM) was polymerized in buffer A containing 1 mM GTP at 37 °C for 15 min in the absence and presence of different concentrations (1–10 µM) of ruthenium red. The polymeric FtsZ was sedimented at 52,000 x g at 30 °C for 20 min. The polymeric mass of FtsZ was determined by subtracting the supernatant concentration from the total protein concentration. The experiment was performed four times.

 
Electron microscopic studies showed an increase in FtsZ protofilaments bundling with an increasing concentration of ruthenium red (Fig. 3). Only a few tiny polymers of FtsZ were observed in the absence of ruthenium red (Fig. 3A), whereas a network of protofilament bundles was detected in the presence of 2 µM ruthenium red (Fig. 3B). More discrete and long protofilaments were visible with 4 µM ruthenium red, indicating a further increase in the protofilament bundling (data not shown). 10 µM of ruthenium red produced a dense network of thick polymers of FtsZ (Fig. 3C). Interestingly, the amount of polymer sedimented in the presence of 4 and 10 µM of ruthenium red were similar; however, 10 µM ruthenium red produced a significantly larger light-scattering signal than that produced by 4 µM ruthenium red. Thus, the increased light-scattering signal was due to extensive bundling of FtsZ polymers in the presence of 10 µM ruthenium red (Fig. 3C). At a relatively high concentration (25 µM), ruthenium red induced extensive aggregation of FtsZ, but filamentous structures were not visible under the conditions used (data not shown). The results of light scattering and electron microscopic studies together demonstrated that FtsZ assembly occurred with a primary assembly of protofilaments followed by ruthenium red-induced bundling.



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  		FIG. 3.
Electron micrographs of FtsZ polymers in the absence and presence of ruthenium red. FtsZ (7.3 µM) was polymerized in buffer A containing different concentrations of ruthenium red in the presence of 1 mM GTP at 37 °C for 10 min. We stained the FtsZ polymers with uranyl acetate as described under "Experimental Procedures." A, electron micrograph of FtsZ polymer in the absence of ruthenium red. B and C, FtsZ polymers in the presence of 2 and 10 µM ruthenium red, respectively. All micrographs were taken at 50,000x magnification. In all cases, error bar = 200 nm.

 
Is Ruthenium Red-induced FtsZ Polymerization GTP-dependent?—FtsZ (7.3 µM) was polymerized in buffer A containing 10 µM of ruthenium red in the absence and presence of 1 mM GTP at 37 °C. There was no significant enhancement of the light scattering in the absence of GTP (Fig. 4). However, in the presence of GTP, light scattering increased to the same extent as that described in Fig. 1, suggesting that GTP was essential for ruthenium red-induced FtsZ polymerization (Fig. 4).



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  		FIG. 4.
Characterization of ruthenium red-induced FtsZ polymerization. FtsZ (7.3 µM) was polymerized in PIPES buffer containing 10 µM ruthenium red in the absence of GTP ({circ}), in the absence of magnesium ({triangleup}), and in the presence of 1 mM GTP and 2 mM magnesium ({blacktriangleup}). The light-scattering signals were corrected by subtracting appropriate blank signals.

 
Ruthenium Red-induced FtsZ Polymerization Requires Magnesium—Because ruthenium red-induced assembly of FtsZ was performed in PIPES buffer containing 2 mM magnesium, we enquired whether magnesium was a requirement for the modulation of FtsZ assembly by ruthenium red. FtsZ was incubated with 10 µM ruthenium red in the absence and presence of 2 mM magnesium on ice. Polymerization reaction was started by adding 1 mM GTP to the reaction mixture and immediately transferring the solution to a cuvette at 37 °C. Ruthenium red produced minimal light-scattering signal in the absence of divalent magnesium, whereas it produced a large light-scattering signal in the presence of 2 mM magnesium (Fig. 4). Further, in the absence of magnesium, only 11% of the total FtsZ was pelleted in the presence of 10 µM ruthenium red. In addition, mostly aggregates of FtsZ and a very few short and thin protofilaments of FtsZ but no bundles were observed in the absence of divalent magnesium by electron microscopy at 50,000x magnification (data not shown). Furthermore, incubation of FtsZ with 10 µM ruthenium red in the absence of both divalent magnesium and GTP produced only aggregates, indicating that GTP hydrolysis was required for the filamentous assembly (data not shown). The GTPase activity of FtsZ was measured in the presence of 10 µM ruthenium red and 1 mM GTP in the absence of divalent magnesium at 37 °C. Ruthenium red did not induce the GTPase activity of FtsZ in the absence of magnesium, suggesting that magnesium was required for promoting the GTPase activity of FtsZ (data not shown). The results together suggested that ruthenium red required divalent magnesium for promoting filamentous assembly of FtsZ and subsequent bundling of FtsZ filaments.

Effect of Ruthenium Red on the GTPase Activity of FtsZ— Ruthenium red reduced the GTPase activity of FtsZ in a concentration-dependent manner (Fig. 5). 2 µM ruthenium red reduced the GTPase activity of FtsZ by 55%, compared with control (in the absence of ruthenium red), and 75% inhibition of the GTPase activity occurred in the presence of 10 µM ruthenium red. Ruthenium red was also found to induce bundling of FtsZ protofilaments, which could be due to the reduction of GTPase activity (Fig. 3). Alternatively, the bundling of protofilaments by ruthenium red could reduce the GTPase activity of FtsZ.



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  		FIG. 5.
Effect of ruthenium red on the GTPase activity of FtsZ. The extent of GTP hydrolysis was measured as described under "Experimental Procedures."

 
Does Ruthenium Red Copolymerize with FtsZ?—FtsZ (7.3 µM) was polymerized with different concentrations (2–10 µM) of ruthenium red, and the polymer-bound ruthenium red molecules were separated from the free ruthenium red molecules by pelleting down the FtsZ polymers. The polymeric pellets of FtsZ were dissolved in PIPES buffer, and the concentrations of ruthenium red and FtsZ were determined as described under "Experimental Procedures." The stoichiometries of incorporation of ruthenium red per FtsZ molecule in the polymer were found to be 0.36 ± 0.01, 0.63 ± 0.13, and 0.82 ± 0.06 in the presence of 2, 6, and 10 µM ruthenium red, respectively (Table I).


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  		TABLE I
Incorporation of ruthenium red in polymeric FtsZ in the absence and presence of 10 mM calcium

 
Comparison of the Effects of Divalent Calcium and Ruthenium Red on the Assembly Properties of FtsZ—We examined the effects of calcium on the polymerization reaction of FtsZ exactly under the same conditions as described previously for ruthenium red and compared it with the effects of ruthenium red on FtsZ polymerization. Similar to previously reported findings (18), our results also showed that millimolar concentrations of divalent calcium enhanced the sedimentable polymer mass in a concentration-dependent manner (Fig. 6). For example, 1 and 5 mM calcium increased the sedimentable polymer mass by 30 and 52%, respectively, compared with the control (Fig. 6). The polymerization reaction reached the optimum at 10 mM calcium. However, ruthenium red exerted similar effects on FtsZ polymer mass at micromolar concentrations (Fig. 2). For instance, 10 mM calcium increased the pelletable polymer mass by 85%, whereas only 10 µM ruthenium red was sufficient to increase the pelletable polymer mass by 98% (Figs. 2 and 6). Thus, divalent calcium required a 1000-fold higher concentration than ruthenium red to produce similar increase in the polymer level.



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  		FIG. 6.
Calcium-induced polymerization of FtsZ. FtsZ (7.3 µM) in buffer A was polymerized in the presence of different concentrations (0–10 mM) of calcium and 1 mM GTP for 15 min at 37 °C. The polymer mass was quantified as described under "Experimental Procedures."

 
Further, we compared the relative potencies of calcium and ruthenium red to prevent dilution-induced disassembly of the preformed FtsZ polymers by a sedimentation assay. FtsZ (30 µM) was polymerized in the presence of 10 mM calcium and 1 mM GTP for 15 min at 37 °C. Under the conditions used, 85 ± 3% of the total FtsZ was pelleted as polymers. However, ~60% of the total protein was pelleted when 7.3 µM FtsZ was used for polymerization (Fig. 6). The results taken together showed that the fraction of total FtsZ pelleted increased at a higher concentration of FtsZ, which is consistent with a previous report (13). The data indicated that the bundling of protofilaments is favored at higher FtsZ concentrations. The preformed FtsZ polymers were diluted 30-fold into warm 25 mM PIPES buffer containing different concentrations of ruthenium red or divalent calcium. After an additional 10 min of incubation either with ruthenium red or divalent calcium at 37 °C, the polymers were sedimented. In the absence of the cations, only 8 ± 3% of the total FtsZ of the diluted polymeric solution was pelleted, indicating that the FtsZ polymers were highly labile. The sedimented protein concentrations in the pellet fractions were found to increase with increasing concentration of ruthenium red (Fig. 7A). For example, 18 ± 5%, 50 ± 6%, and 70 ± 3% of the total FtsZ were pelleted in the presence of 1, 2, and 10 µM of ruthenium red, respectively. Thus, ruthenium red strongly stabilized FtsZ polymers against dilution-induced disassembly. Like ruthenium red, divalent calcium also increased the pelletable polymer mass in a concentration-dependent manner (Fig. 7B). For example, 19 ± 5% of total FtsZ was pelleted in the presence of 5 mM calcium, and 52 ± 2% FtsZ was pelleted in the presence of 10 mM calcium. Thus, similar to ruthenium red, calcium also stabilized FtsZ polymers against dilution-induced disassembly; however, calcium required a 1000-fold higher concentration than ruthenium red to produce similar stabilizing effects upon FtsZ polymers.



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  		FIG. 7.
Stabilization of FtsZ polymers against dilution-induced disassembly. A and B, the fraction of total FtsZ pelleted in the presence of different concentrations of ruthenium red and calcium, respectively. The fraction of total protein pelleted was determined as described in Fig. 2. The data represent an average of four individual experiments.

 
Does Ruthenium Red Bind at the Calcium Binding Site(s) of FtsZ?—We used two approaches to find out whether ruthenium red shares its binding site on FtsZ with calcium. In the first approach, FtsZ was polymerized in the presence of ruthenium red with or without 10 mM divalent calcium. The polymeric FtsZ was sedimented down, and the stoichiometry of ruthenium red incorporation per polymeric FtsZ was determined by measuring the concentrations of FtsZ and ruthenium red in the pellets. Calcium reduced the stoichiometry of ruthenium red incorporation in polymeric FtsZ by 76%, from 0.82 to 0.2 (Table I). In the second approach, FtsZ was incubated with 10 µM ruthenium red without GTP for 30 min in the absence and presence of 10 mM divalent calcium at 0 °C. The stoichiometry of ruthenium red incorporated per soluble FtsZ was determined after gel filtration and exhaustive dialysis. The stoichiometries of incorporation of ruthenium red per monomer of FtsZ were found to be 0.7 and 0.3 in the absence and presence of 10 mM calcium, respectively. Calcium also displaced ruthenium red from the preformed FtsZ-ruthenium red complex. For example, the stoichiometry of ruthenium red incorporation per FtsZ was reduced by 55% by 10 mM calcium. The results together suggested that ruthenium red and calcium share their binding sites on FtsZ, and that ruthenium red could be used as a probe to determine the binding site of calcium on FtsZ and the role of calcium on FtsZ assembly (29, 30).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The intensity of light scattering depends upon the polymer mass as well as the size and shape of the polymers (36). Under the conditions used in this study, FtsZ formed small single protofilaments in the absence of ruthenium red, and these thin polymers did not scatter light efficiently. Ruthenium red increased the light-scattering signal of FtsZ assembly in a biphasic manner, suggesting that the assembly of FtsZ occurred in two steps in the presence of ruthenium red. The first step involved the assembly of FtsZ into protofilaments, and then ruthenium red induced the bundling of single protofilaments that scattered light intensely. The bundling of protofilaments increased the size of the polymers that not only assisted the visualization of polymers by electron microscopy, but also facilitated efficient pelleting of protofilaments, which were otherwise difficult to pellet. For example, even using 280,000 x g, only 27% of the total FtsZ was sedimented in the presence of 2.5 mM magnesium, whereas 50 and 51% of the total FtsZ were sedimented in the presence of 10 mM magnesium (12, 22). Alternatively, small single protofilaments of FtsZ that were formed in the absence of ruthenium red or calcium disassembled during the centrifugation. The cations prevented depolymerization of the protofilaments by stabilizing the polymers, which, in turn, increased the pelleted polymeric mass. Therefore, although it seemed that ruthenium red and calcium increased the polymeric mass of FtsZ, the actual polymeric mass, i.e. assembled protofilaments, might be nearly the same with or without the cations. However, we could not rule out the possibility that the observed increase in the polymer mass in the presence of ruthenium red was at least partly due to the promotion of FtsZ assembly.

The polycation DEAE-dextran and lipid monolayer have been shown to assemble FtsZ into large protofilaments, sheets, and minirings in the absence of GTP (11). In contrast to the stimulating effects of DEAE-dextran on both FtsZ and microtubule polymerization, ruthenium red promoted FtsZ assembly, but it inhibited microtubule polymerization (34, 37). DEAE-dextran has multiple positive charges, and it is an extremely large molecule (molecular weight = 500,000), ~12 times bigger in size than FtsZ. Thus, it is reasonable to consider that the binding sites of DEAE-dextran and ruthenium red on FtsZ are also different. Further, ruthenium red needed GTP and magnesium to induce FtsZ assembly, whereas DEAE-dextran promotes assembly in the absence of GTP, suggesting that DEAE-dextran and ruthenium red induce FtsZ assembly by different mechanisms.

Ruthenium red completely inhibits microtubule assembly at stoichiometric concentrations and binds to tubulin tightly (33, 34). Ward et al. (38) suggested that ruthenium red binds to the negatively charged carboxyl-terminal domain of the {alpha}-tubulin. Ruthenium red did not bind to the calcium-binding site on tubulin, suggesting that they have different binding sites on tubulin (33, 34). However, calcium and ruthenium red disassembled microtubules and inhibited microtubule polymerization (33, 34). Surprisingly, both calcium and ruthenium red promoted FtsZ assembly in a similar fashion. The differential behavior of calcium and ruthenium red toward the polymerization of tubulin and FtsZ might be due to the different binding interactions of these metal ions with tubulin and FtsZ. Like tubulin, FtsZ also has a negatively charged carboxyl-terminal tail. Ma and Margolin (39) found that the C-terminal-truncated FtsZ polymerized in the presence of calcium and suggested that calcium has a binding site on FtsZ which is not located at the truncated C-terminal. Thus, the binding site of calcium on FtsZ seems to be different from its binding site on tubulin. We found that calcium inhibited the binding of ruthenium red to FtsZ, suggesting that ruthenium red shares its binding site on FtsZ with calcium. Therefore, it is possible that, similar to calcium, ruthenium red also binds to a site on FtsZ that is not located at the C terminus.

Substoichiometric incorporation of ruthenium red into FtsZ polymers strongly reduced the GTP hydrolysis rate and stabilized the polymers against disassembly (Table I; Figs. 5 and 7). The bundling of FtsZ polymers could be due to an increase in the lateral interaction among the protofilaments in the presence of ruthenium red. Increased lateral interaction among the protofilaments reduced the solvent-accessible surface area around the nucleotide-binding site on FtsZ that was thought to inhibit the rate of subunit exchange (40). Thus, the reduced GTPase activity of FtsZ could be due to a strong stabilization of FtsZ polymers in the presence of ruthenium red. Alternatively, the reduced GTPase activity could increase the stability of the polymers in the presence of ruthenium red.

The strong effects of ruthenium red on FtsZ assembly indicated the presence of an important regulatory site on FtsZ. It has been hypothesized that carefully balanced polymer stability plays an important role in the positional regulation of FtsZ. It is conceivable that, similar to the fine regulation of actin and microtubule polymerization by their accessory proteins, FtsZ assembly is also finely regulated by its associated proteins. A few proteins have already been identified that regulate FtsZ assembly and its functional organization (1, 2, 24–26). For example, it has been shown that a basic protein ZipA promotes and stabilizes protofilament assembly of FtsZ (25, 26). Further, ZipA assists to arrange FtsZ protofilaments into arrays of long bundles or sheets and plays a critical role in the organization of the FtsZ ring in bacterial cells (25, 26). Thus, it is tempting to speculate that ruthenium red may mimic the action of a positively charged peptide or a protein that regulates the assembly dynamics of FtsZ in bacteria.


   FOOTNOTES
 
* This study was funded by a grant from the Department of Science and Technology, Government of India (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a fellowship from the Council of Scientific and Industrial Research, Government of India. Back

§ To whom correspondence should be addressed. Tel.: 91-22-2576-7838; Fax: 91-22-2572-3480; E-mail: panda{at}iitb.ac.in.

1 The abbreviations used are: GTP, guanosine 5'-triphosphate; PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); BSA, bovine serum albumin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. P. Erickson for providing us with the FtsZ clone, and Drs. T. R. S. Prasanna, S. Patankar, and D. Dasgupta for critical reading of the manuscript. We also thank the Regional Sophisticated Instrumentation Centre, Indian Institute of Technology, Bombay for allowing us to use the electron microscopy facility.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Errington, J., Daniel, R. A., and Scheffers, D. J. (2003) Microbiol. Mol. Biol. Rev. 67, 52–65[Abstract/Free Full Text]
  2. Wang, X., and Lutkenhaus, J. (1996) Mol. Microbiol. 21, 313–319[CrossRef][Medline] [Order article via Infotrieve]
  3. Stricker, J., Maddox, P., Salmon, E. D., and Erickson, H. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3171–3175[Abstract/Free Full Text]
  4. Rueda, S., Vicente, M., and Mingorance, J. (2003) J. Bacteriol. 185, 3344–3351[Abstract/Free Full Text]
  5. Lutkenhaus, J. (2002) Dev. Cell. 2, 519–521[Medline] [Order article via Infotrieve]
  6. Rothfield, L., Justice, S., and Garcia-Lara, J. (1999) Annu. Rev. Genet. 33, 423–448[CrossRef][Medline] [Order article via Infotrieve]
  7. Lutkenhaus, J., and Addinall, S. G. (1997) Annu. Rev. Biochem. 66, 93–116[CrossRef][Medline] [Order article via Infotrieve]
  8. Lowe, J., and Amos, L. A. (1998) Nature 391, 203–206[CrossRef][Medline] [Order article via Infotrieve]
  9. Nogales, E., Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199–203[CrossRef][Medline] [Order article via Infotrieve]
  10. Erickson, H. P. (1995) Cell 80, 367–370[CrossRef][Medline] [Order article via Infotrieve]
  11. Nogales, E., Downing, K. H., Amos, L. A., and Lowe, J. (1998) Nat. Struct. Biol. 5, 451–458[CrossRef][Medline] [Order article via Infotrieve]
  12. Bramhill, D., and Thompson, C. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5813–5817[Abstract/Free Full Text]
  13. Mukherjee, A., and Lutkenhaus, J. (1998) EMBO J. 17, 462–469[CrossRef][Medline] [Order article via Infotrieve]
  14. Lu, C., Reedy, M., and Erickson, H. P. (2000) J. Bacteriol. 182, 164–170[Abstract/Free Full Text]
  15. Lu, C., Stricker, J., and Erickson, H. P. (1998) Cell Motil. Cytoskeleton 40, 71–86[CrossRef][Medline] [Order article via Infotrieve]
  16. Romberg, L., Simon, M., and Erickson, H. P. (2001) J. Biol. Chem. 276, 11743–11753[Abstract/Free Full Text]
  17. Erickson, H. P., Taylor, D. W., Taylor, K. A., and Bramhill, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 519–523[Abstract/Free Full Text]
  18. Yu, X. C., and Margolin, W. (1997) EMBO J. 16, 5455–5463[CrossRef][Medline] [Order article via Infotrieve]
  19. Beuria, T. K., Krishnakumar, S. S., Sahar, S., Singh, N., Gupta, K., Meshram, M., and Panda D. (2003) J. Biol. Chem. 278, 3735–3741[Abstract/Free Full Text]
  20. Small, E., and Addinall, S. G. (2003) Microbiology 149, 2235–2242[Abstract/Free Full Text]
  21. Rivas, G., Lopez, A., Mingorance, J., Ferrandiz, M. J., Zorrilla, S., Minton, A. P., Vicente, M., and Andreu, J. M. (2000) J. Biol. Chem. 275, 11740–11749[Abstract/Free Full Text]
  22. Mukherjee, A., and Lutkenhaus, J. (1999) J. Bacteriol. 181, 823–832[Abstract/Free Full Text]
  23. Gonzalez, J. M., Jimenez, M., Velez, M., Mingorance, J., Andreu, J. M., Vicente, M., and Rivas, G. (2003) J. Biol. Chem. 278, 37664–37671[Abstract/Free Full Text]
  24. Hale, C. A., and de Boer, P. A. (1997) Cell 88, 175–185[CrossRef][Medline] [Order article via Infotrieve]
  25. RayChaudhuri, D. (1999) EMBO J. 18, 2372–2383[CrossRef][Medline] [Order article via Infotrieve]
  26. Hale, C. A., Rhee, A. C., and de Boer, P. A. (2000) J. Bacteriol. 182, 5153–5166[Abstract/Free Full Text]
  27. Margolin W. (2003) Curr. Biol. 13, R16–R18[CrossRef][Medline] [Order article via Infotrieve]
  28. Koppelman, C. M., Aarsman, M. E., Postmus, J., Pas, E., Muijsers, A. O., Scheffers, D. J., Nanninga, N., and den Blaauwen, T. (2004) Mol. Microbiol. 51, 645–657[CrossRef][Medline] [Order article via Infotrieve]
  29. Sasaki, T., Naka, M., Nakamura, F., and Tanaka, T. (1992) J. Biol. Chem. 267, 21518–21523[Abstract/Free Full Text]
  30. Yamada, A., Sato, O., Watanabe, M., Walsh, M. P., Ogawa, Y., and Imaizumi, Y. (2000) Biochem. J. 349, 797–804[Medline] [Order article via Infotrieve]
  31. Nakamura, F., Naka, M., and Tanaka, T. (1992) FEBS Lett. 314, 93–96[CrossRef][Medline] [Order article via Infotrieve]
  32. Chamberlain, B. K., Volpe, P., and Fleischer, S. (1984) J. Biol. Chem. 259, 7547–7553[Abstract/Free Full Text]
  33. Deinum, J., Wallin, M., and Jensen, P. W. (1985) Biochim. Biophys. Acta 838, 197–205[Medline] [Order article via Infotrieve]
  34. Deinum, J., Wallin, M., Kanje, M., and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209–213[Medline] [Order article via Infotrieve]
  35. Santra, M. K., and Panda, D. (2003) J. Biol. Chem. 278, 21336–21343[Abstract/Free Full Text]
  36. Gaskin, F., Cantor, C. R., and Shelanski, M. L. (1974) J. Mol. Biol. 89, 737–755[CrossRef][Medline] [Order article via Infotrieve]
  37. Erickson, H. P., and Voter, W. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2813–2817[Abstract/Free Full Text]
  38. Ward, L. D., Seckler, R., and Timasheff, S. N. (1994) Biochemistry 33, 11900–11908[CrossRef][Medline] [Order article via Infotrieve]
  39. Ma, X., and Margolin, W. (1999) J. Bacteriol. 181, 7531–7544[Abstract/Free Full Text]
  40. Mingorance, J., Rueda, S., Gomez-Puertas, P., Valencia, A., and Vicente, M. (2001) Mol. Microbiol. 41, 83–91[CrossRef][Medline] [Order article via Infotrieve]
  41. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]

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