The pH dependence of polymerization and bundling by the essential bacterial cytoskeletal protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)--it stabilizes associations of protofilaments.     

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.     

Characterization of Caulobacter crescentus FtsZ protein using dynamic light scattering

The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.     

Analysis of FtsZ Assembly by Light Scattering and Determination of the Role of Divalent Metal Cations

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90° angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg²⁺ was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca²⁺ mediated dynamic assembly of FtsZ (X.-C. Yu and W. Margolin, EMBO J. 16:5455–5463, 1997). However, we found that Ca²⁺ was not required for FtsZ assembly and that this concentration of Ca²⁺ reduced the dynamic behavior of FtsZ assembly.     

AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ

AAA⁺ chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers.     

Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.     

Membrane reconstitution of FtsZ–ZipA complex inside giant spherical vesicles made of E. coli lipids: Large membrane dilation and analysis of membrane plasticity

During the division process of Escherichia coli, the globular protein FtsZ is early recruited at the constriction site. The Z-ring, based on FtsZ filaments associated to the inner cell membrane, has been postulated to exert constriction forces. Membrane anchoring is mediated by ZipA, an essential transmembrane protein able to specifically bind FtsZ. In this work, an artificial complex of FtsZ–ZipA has been reconstituted at the inner side of spherical giant unilamellar vesicles made of E. coli lipids. Under these conditions, FtsZ polymerization, triggered when a caged GTP analogue is UV-irradiated, was followed by up to 40% vesicle inflation. The homogeneous membrane dilation was accompanied by the visualization of discrete FtsZ assemblies at the membrane. Complementary rheological data revealed enhanced elasticity under lateral dilation. This explains why vesicles can undergo large dilations in the regime of mechanical stability. A mechanical role for FtsZ polymers as promoters of membrane softening and plasticization is hypothesized.     

The Nucleoid Occlusion SlmA Protein Accelerates the Disassembly of the FtsZ Protein Polymers without Affecting Their GTPase Activity

Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ.     

Glutamate-induced assembly of bacterial cell division protein FtsZ

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; approximately 90% of the protein was sedimented as polymer in the presence of 1 m glutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 m glutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.     

Structural and Functional Model for Ionic (K/Na) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP-Mg²⁺ and monovalent cations. The presence of a K⁺ ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K⁺. This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na⁺ destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.     

Slow polymerization of Mycobacterium tuberculosis FtsZ

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.     

Location of Dual Sites in E. coli FtsZ Important for Degradation by ClpXP; One at the C-Terminus and One in the Disordered Linker

ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One substrate degraded by Escherichia coli ClpXP is FtsZ, an essential cell division protein. FtsZ forms polymers that assemble into a large ring-like structure, termed the Z-ring, during cell division at the site of constriction. The FtsZ monomer is composed of an N-terminal polymerization domain, an unstructured linker region and a C-terminal conserved region. To better understand substrate selection by ClpXP, we engineered FtsZ mutant proteins containing amino acid substitutions or deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for degradation of both FtsZ monomers and polymers by ClpXP in vitro. One region is located 30 residues away from the C-terminus in the unstructured linker region that connects the polymerization domain to the C-terminal region. The other region is near the FtsZ C-terminus and partially overlaps the recognition sites for several other FtsZ-interacting proteins, including MinC, ZipA and FtsA. Mutation of either region caused the protein to be more stable and mutation of both caused an additive effect, suggesting that both regions are important. We also observed that in vitro MinC inhibits degradation of FtsZ by ClpXP, suggesting that some of the same residues in the C-terminal site that are important for degradation by ClpXP are important for binding MinC.     

Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments

Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.     

Behaviour of bacterial division protein FtsZ under a monolayer with phospholipid domains

Assembly of the tubulin-like protein FtsZ at or near the cytoplasmic membrane is one of the earliest steps in division of bacteria such as Escherichia coli. Exactly what constitutes the site at which FtsZ acts is less clear. To investigate the influence of the membrane phospholipids on FtsZ localization and assembly, we have elaborated with the Langmuir technique a two-lipid monolayer made of dilauryl-phosphatidylethanolamine (DLPE) and dipalmitoyl-phosphatidylglycerol (DPPG). This monolayer comprised stable condensed domains in an expanded continuous phase. In the presence of GTP, FtsZ assembly disrupts the condensed domains within 5 min. After several hours, with or without GTP, FtsZ assembled into large aggregates at the domain interface. We suggest that the GTP-induced polymerization of FtsZ is coupled to the association of FtsZ protofilaments with domain interfaces.     

Molecular dynamics simulation of GTPase activity in polymers of the cell division protein FtsZ

FtsZ, the prokaryotic ortholog of tubulin, assembles into polymers in the bacterial division ring. The interfaces between monomers contain a GTP molecule, but the relationship between polymerization and GTPase activity is not unequivocally proven. A set of short FtsZ polymers were modelled and the formation of active GTPase structures was monitored using molecular dynamics. Only the interfaces nearest the polymer ends exhibited an adequate geometry for GTP hydrolysis. Simulated conversion of interfaces from close-to-end to internal position and vice versa resulted in their spontaneous rearrangement between active and inactive conformations. This predicted behavior of FtsZ polymer ends was supported by in vitro experiments.     

The interactions of cell division protein FtsZ with guanine nucleotides

Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.     

Mg(2+)-Linked Self-Assembly of FtsZ in the Presence of GTP or a GTP Analogue Involves the Concerted Formation of a Narrow Size Distribution of Oligomeric Species

The assembly of the bacterial cell division FtsZ protein in the presence of constantly replenished GTP was studied as a function of Mg(2+) concentration (at neutral pH and 0.5 M potassium) under steady-state conditions by sedimentation velocity, concentration-gradient light scattering, fluorescence correlation spectroscopy, and dynamic light scattering. Sedimentation velocity measurements confirmed previous results indicating cooperative appearance of a narrow size distribution of finite oligomers with increasing protein concentration. The concentration dependence of light scattering and diffusion coefficients independently verified the cooperative appearance of a narrow distribution of high molecular weight oligomers, and in addition provided a measurement of the average size of these species, which corresponds to 100 ± 20 FtsZ protomers at millimolar Mg(2+) concentration. Parallel experiments on solutions containing guanosine-5'-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), a slowly hydrolyzable analogue of GTP, in place of GTP, likewise indicated the concerted formation of a narrow size distribution of fibrillar oligomers with a larger average mass (corresponding to 160 ± 20 FtsZ monomers). The closely similar behavior of FtsZ in the presence of both GTP and GMPCPP suggests that the observations reflect equilibrium rather than nonequilibrium steady-state properties of both solutions and exhibit parallel manifestations of a common association scheme.     

Structural Change in FtsZ Induced by Intermolecular Interactions between Bound GTP and the T7 Loop

FtsZ is a prokaryotic homolog of tubulin and is a key molecule in bacterial cell division. FtsZ with bound GTP polymerizes into tubulin-like protofilaments. Upon polymerization, the T7 loop of one subunit is inserted into the nucleotide-binding pocket of the second subunit, which results in GTP hydrolysis. Thus, the T7 loop is important for both polymerization and hydrolysis in the tubulin/FtsZ family. Although x-ray crystallography revealed both straight and curved conformations of tubulin, only a curved structure was known for FtsZ. Recently, however, FtsZ from Staphylococcus aureus has been shown to have a very different conformation from the canonical FtsZ structure. The present study was performed to investigate the structure of FtsZ from Staphylococcus aureus by mutagenesis experiments; the effects of amino acid changes in the T7 loop on the structure as well as on GTPase activity were studied. These analyses indicated that FtsZ changes its conformation suitable for polymerization and GTP hydrolysis by movement between N- and C-subdomains via intermolecular interactions between bound nucleotide and residues in the T7 loop.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

A rapid fluorescence assay for FtsZ assembly indicates cooperative assembly with a dimer nucleus

FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?