FtsZ in Bacterial Cytokinesis: Cytoskeleton and Force Generator All in One

Summary: FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation.     

In vivo organization of the FtsZ‐ring by ZapA and ZapB revealed by quantitative super‐resolution microscopy

In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring‐like structure (Z‐ring) at the midcell. Despite its essential role, the molecular architecture of the Z‐ring remains elusive. In this work we examine the roles of two FtsZ‐associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z‐ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single‐molecule‐based super‐resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z‐ring. Furthermore, we find these clusters are not due to the loss of ZapB–MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.     

Large ring polymers align FtsZ polymers for normal septum formation

Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria. Mutation of sepF results in deformed division septa. It is unclear how SepF contributes to the synthesis of normal septa. We have studied SepF by electron microscopy (EM) and found that the protein assembles into very large (～50 nm diameter) rings. These rings were able to bundle FtsZ protofilaments into strikingly long and regular tubular structures reminiscent of eukaryotic microtubules. SepF mutants that disturb interaction with FtsZ or that impair ring formation are no longer able to align FtsZ filaments in vitro, and fail to support normal cell division in vivo. We propose that SepF rings are required for the regular arrangement of FtsZ filaments. Absence of this ordered state could explain the grossly distorted septal morphologies seen in sepF mutants.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.     

Investigation of regulation of FtsZ assembly by SulA and development of a model for FtsZ polymerization

In Escherichia coli FtsZ organizes into a cytoskeletal ring structure, the Z ring, which effects cell division. FtsZ is a GTPase, but the free energy of GTP hydrolysis does not appear to be used for generation of the constriction force, leaving open the question of the function of the GTPase activity of FtsZ. Here we study the mechanism by which SulA, an inhibitor of FtsZ induced during the SOS response, inhibits FtsZ function. We studied the effects of SulA on the in vitro activities of FtsZ, on Z rings in vivo, and on a kinetic model for FtsZ polymerization in silico. We found that the binding of SulA to FtsZ is necessary but not sufficient for inhibition of polymerization, since the assembly of FtsZ polymers in the absence of the GTPase activity was not inhibited by SulA. We developed a new model for FtsZ polymerization that accounts for the cooperativity of FtsZ and could account for cooperativity observed in other linear polymers. When SulA was included in the kinetic scheme, simulations revealed that SulA with strong affinity for FtsZ delayed, but did not prevent, the assembly of polymers when they were not hydrolyzing GTP. Furthermore, the simulations indicated that SulA controls the assembly of FtsZ by binding to a polymerization-competent form of the FtsZ molecule and preventing it from participating in assembly. In vivo stoichiometry of the disruption of Z rings by SulA suggests that FtsZ may undergo two cooperative transitions in forming the Z ring.     

FtsA reshapes membrane architecture and remodels the Z‐ring in Escherichia coli

Cell division in prokaryotes initiates with assembly of the Z‐ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z‐ring is comprised of polymers of tubulin‐like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C‐terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide‐dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C‐terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z‐ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.     

Localization of division protein FtsZ in Mycoplasma hominis

The localization of FtsZ protein in M. hominis cells was studied by immunoelectron microscopy with polyclonal antibodies to this protein. Cell polymorphism typical for mycoplasmas was seen on electron microscopic pictures. Among the diversity of cell shapes, we distinguished dumbbell-shaped dividing cells and cells connected with each other by membrane tubules (former constrictions). The label was predominantly observed in the constriction area of dividing M. hominis cells and on thin membrane tubules. A septum and the gold labeling of this structure have not been described before in mycoplasma cells. For the first time, in some rounded and oval cells, colloidal gold particles labeled the entire plasma membrane, probably marking a submembranous contractile ring (Z ring). These observations confirm the implication of FtsZ protein in M. hominis cytokinesis. In some cells, the spiral-like distribution of gold particles was observed. Most likely, FtsZ protofilaments in M. hominis cells form spiral structures similar to Z spirals in Bacillus subtilis and Escherichia coli. Their presence in mycoplasma cells may be considered to be an important argument in favor of Z ring assembly through the reorganization of Z spirals. FtsZ as a bacterial cytoskeleton protein binding with membrane directly or through intermediates may be engaged in maintenance of M. hominis cell shape.     

Curved FtsZ protofilaments generate bending forces on liposome membranes

We have created FtsZ‐YFP‐mts where an amphipathic helix on the C‐terminus tethers FtsZ to the membrane. When incorporated inside multi‐lamellar tubular liposomes, FtsZ‐YFP‐mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ‐YFP‐mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane‐targeting sequence (mts) from the C‐terminus to the N‐terminus, which is approximately 180 degrees from the C‐terminal tether. When mts‐FtsZ‐YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending.     

Temperature shift experiments with an ftsZ84(Ts) strain reveal rapid dynamics of FtsZ localization and indicate that the Z ring is required throughout septation and cannot reoccupy division sites once constriction has initiated.

FtsZ is an essential division protein in bacteria that functions by forming a ring at midcell that mediates septation. To further study the function of the Z ring the effect of a temperature-sensitive mutation, ftsZ84(Ts), on ring dynamics and septal progression was examined. Shifting a strain carrying an ftsZ84(Ts) mutation to the nonpermissive temperature led to loss of Z rings within 1 min. Septal ingrowth was immediately inhibited, and sharply demarcated septa, present at the time of the shift, were gradually replaced by blunted septa. These results indicate that the Z ring is required throughout septation. Shifting filaments to permissive temperature led to a rapid localization of FtsZ84 at regular intervals. Included in these localization events were complete and partial rings as well as spots, although some of these eventually aborted. These results reveal the rapid dynamics of FtsZ localization and indicate that nucleation sites are formed in the absence of FtsZ function. Interestingly, Z rings could not reform at division sites that were constricted although they could reform at sites that had not begun constriction.     

Inside-out Z rings--constriction with and without GTP hydrolysis

The bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled 'inside-out' Z rings that wrapped around the outside surface of tubular liposomes. The inside-out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z-ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.     

FtsZ condensates: An in vitro electron microscopy study

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto‐filaments. Although FtsZ assembles into single proto‐filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z‐ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 340–350, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) undergoes at least two kinds of cell division: vegetative septation leading to cross-walls in the substrate mycelium; and developmentally regulated sporulation septation in aerial hyphae. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrate a difference between the two types of septation. The ftsZ17(Spo) allele gave rise to a classical white phenotype. The mutant grew as well as the parent on plates, and formed apparently normal hyphal cross-walls, although with a small reduction in frequency. In contrast, sporulation septation was almost completely abolished, resulting in a phenotype reminiscent of whiH and ftsZdelta2p mutants. The ftsZ17(Spo) allele was partially dominant and had no detectable effect on the cellular FtsZ content. As judged from both immunofluorescence microscopy of FtsZ and translational fusion of ftsZ to egfp, the mutation prevented correct temporal and spatial assembly of Z rings in sporulating hyphae. Homology modelling of S. coelicolor FtsZ indicated that the mutation, an A249T change in the C-terminal domain, would be expected to alter the protein on the lateral face of FtsZ protofilaments. The results suggest that cytokinesis may be developmentally controlled at the level of Z-ring assembly during sporulation of S. coelicolor A3(2).     

FtsZ dynamics in bacterial division: What,how, and why?

Bacterial cell division is orchestrated by the divisome, a protein complex centered on the tubulin homolog FtsZ. FtsZ polymerizes into a dynamic ring that defines the division site, recruits downstream proteins, and directs peptidoglycan synthesis to drive constriction. Recent studies have documented treadmilling of FtsZ polymer clusters both in cells and in vitro. Emerging evidence suggests that FtsZ dynamics are regulated largely by intrinsic properties of FtsZ itself and by the membrane anchoring protein FtsA. Although FtsZ dynamics are broadly required for Z-ring assembly, their role(s) during constriction may vary among bacterial species. These recent advances set the stage for future studies to investigate how FtsZ dynamics are physically and/or functionally coupled to peptidoglycan metabolic enzymes to direct efficient division.     

Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.     

Chloroplast Division Protein ARC3 Regulates Chloroplast FtsZ-Ring Assembly and Positioning in Arabidopsis through Interaction with FtsZ2

Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid.     

A conserved coiled‐coil protein pair focuses the cytokinetic Z‐ring in Caulobacter crescentus

The cytoskeletal GTPase FtsZ assembles at midcell, recruits the division machinery and directs envelope invagination for bacterial cytokinesis. ZapA, a conserved FtsZ‐binding protein, promotes Z‐ring stability and efficient division through a mechanism that is not fully understood. Here, we investigated the function of ZapA in Caulobacter crescentus. We found that ZapA is encoded in an operon with a small coiled‐coil protein we named ZauP. ZapA and ZauP co‐localized at the division site and were each required for efficient division. ZapA interacted directly with both FtsZ and ZauP. Neither ZapA nor ZauP influenced FtsZ dynamics or bundling, in vitro, however. Z‐rings were diffuse in cells lacking zapA or zauP and, conversely, FtsZ was enriched at midcell in cells overproducing ZapA and ZauP. Additionally, FtsZ persisted at the poles longer when ZapA and ZauP were overproduced, and frequently colocalized with MipZ, a negative regulator of FtsZ polymerization. We propose that ZapA and ZauP promote efficient cytokinesis by stabilizing the midcell Z‐ring through a bundling‐independent mechanism. The zauPzapA operon is present in diverse Gram‐negative bacteria, indicating a common mechanism for Z‐ring assembly.     

Synthetic developmental regulator MciZ targets FtsZ across Bacillus species and inhibits bacterial division

Cell division in most bacteria is directed by FtsZ, a conserved tubulin‐like GTPase that assembles forming the cytokinetic Z‐ring and constitutes a target for the discovery of new antibiotics. The developmental regulator MciZ, a 40‐amino acid peptide endogenously produced during Bacillus subtilis sporulation, halts cytokinesis in the mother cell by inhibiting FtsZ. The crystal structure of a FtsZ:MciZ complex revealed that bound MciZ extends the C‐terminal β‐sheet of FtsZ blocking its assembly interface. Here we demonstrate that exogenously added MciZ specifically inhibits B. subtilis cell division, sporulation and germination, and provide insight into MciZ molecular recognition by FtsZ from different bacteria. MciZ and FtsZ form a complex with sub‐micromolar affinity, analyzed by analytical ultracentrifugation, laser biolayer interferometry and isothermal titration calorimetry. Synthetic MciZ analogs, carrying single amino acid substitutions impairing MciZ β‐strand formation or hydrogen bonding to FtsZ, show a gradual reduction in affinity that resembles their impaired activity in bacteria. Gene sequences encoding MciZ spread across genus Bacillus and synthetic MciZ slows down cell division in Bacillus species, including pathogenic Bacillus cereus and Bacillus anthracis. Moreover, B. subtilis MciZ is recognized by the homologous FtsZ from Staphylococcus aureus and inhibits division when it is expressed into S. aureus cells.     

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus

In most bacteria, the tubulin‐like GTPase FtsZ forms an annulus at midcell (the Z‐ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z‐ring assembly and early FtsZ‐directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C‐terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane‐anchored FtsZ in the regulation of cell wall hydrolysis.     

Division‐site localization of RodZ is required for efficient Z ring formation in Escherichia coli

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ‐dependent manner. We also found that division‐site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division‐site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.