FtsZ Filaments Research Articles

Self-Organization of FtsZ Polymers in Solution Reveals Spacer Role of the Disordered C-Terminal Tail

FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5–5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit −220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (∼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.

Z-ring Structure and Constriction Dynamics in E. coli.

The Z-ring plays a central role in bacterial division. It consists of FtsZ filaments, but the way these reorganize in the ring-like structure during septation remains largely unknown. Here, we measure the effective constriction dynamics of the ring. Using an oscillating optical trap, we can switch individual rod-shaped E. coli cells between horizontal and vertical orientations. In the vertical orientation, the fluorescent Z-ring image appears as a symmetric circular structure that renders itself to quantitative analysis. In the horizontal orientation, we use phase-contrast imaging to determine the extent of the cell constriction and obtain the effective time of division. We find evidence that the Z-ring constricts at a faster rate than the cell envelope such that its radial width (inwards from the cytoplasmic membrane) grows during septation. In this respect, our results differ from those recently obtained using photoactivated localization microscopy (PALM) where the radial width of the Z-ring was found to be approximately constant as the ring constricts. A possible reason for the different behavior of the constricting Z-rings could be the significant difference in the corresponding cell growth rates.

Mutations on FtsZ lateral helix H3 that disrupt cell viability hamper reorganization of polymers on lipid surfaces

FtsZ filaments localize at the middle of the bacterial cell and participate in the formation of a contractile ring responsible for cell division. Previous studies demonstrated that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the lateral helix H3 bend of Escherichia coli FtsZ are required for in vivo cell division. In order to understand how these lateral mutations impair the formation of a contractile ring,we extend previous in vitro characterization of these mutants in solution to study their behavior on lipid modified surfaces. We study their interaction with ZipAand look at their reorganization on the surface. We found that the dynamic bundling capacity of the mutant proteins is deficient, and this impairment increases the more the composition and spatial arrangement of the reconstituted system resembles the situation inside the cell: mutant proteins completely fail to reorganize to form higher order aggregates when bound to an E.coli lipid surface through oriented ZipA.We conclude that these surface lateral point mutations affect the dynamic reorganization of FtsZ filaments into bundles on the cell membrane, suggesting that this event is relevant for generating force and completing bacterial division.

ZipA and FtsA* stabilize FtsZ-GDP miniring structures

The cytokinetic division ring of Escherichia coli comprises filaments of FtsZ tethered to the membrane by FtsA and ZipA. Previous results suggested that ZipA is a Z-ring stabilizer, since in vitro experiments it is shown that ZipA enhanced FtsZ assembly and caused the filaments to bundles. However, this function of ZipA has been challenged by recent studies. First, ZipA-induced FtsZ bundling was not significant at pH greater than 7. Second, some FtsA mutants, such as FtsA* were able to bypass the need of ZipA. We reinvestigated the interaction of FtsZ with ZipA in vitro. We found that ZipA not only stabilized and bundled straight filaments of FtsZ-GTP, but also stabilized the highly curved filaments and miniring structures formed by FtsZ-GDP. FtsA* had a similar stabilization of FtsZ-GDP minirings. Our results suggest that ZipA and FtsA* may contribute to constriction by stabilizing this miniring conformation.

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis.

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.

Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division.

The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.

GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis.

The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.

Chiral Vortex Dynamics on Membranes is an Intrinsic Property of FtsZ Driven by GTP Hydrolysis

The primary protein of the bacterial Z ring guiding cell division, FtsZ, has recently been shown to engage in intriguing self-organization together with one of its natural membrane anchors, FtsA. When co-reconstituted on flat supported membranes, these proteins assemble into dynamic chiral vortices whose diameters resemble the cell circumference. These dynamics are due to treadmilling polar FtsZ filaments, supposedly destabilized by the co-polymerizing membrane adaptor FtsA, thus catalysing their turnover. Here we show that FtsA is in fact dispensable and that the phenomenon is an intrinsic property of FtsZ alone when supplemented with a membrane anchor. The emergence of these chiral dynamic patterns occurs at intermediate FtsZ surface densities, in agreement with theoretical predictions, and beyond a threshold GTP concentration. The interplay of membrane tethering, GTP binding, and hydrolysis promotes both, the assembly and the destabilization of FtsZ polymers, leading to the observed treadmilling dynamics. Notably, the vortex chirality is defined by the position of the membrane targeting sequence (mts) and can be inverted when attaching it to the opposite end of FtsZ. This reveals the vectorial character of the filament-supported membrane system consisting of three orthogonal directions, filament polarity, curvature, and membrane attachment.

E. coli Cell Cycle Machinery.

Cytokinesis in E. coli is organized by a cytoskeletal element designated the Z ring. The Z ring is formed at midcell by the coalescence of FtsZ filaments tethered to the membrane by interaction of FtsZ's conserved C-terminal peptide (CCTP) with two membrane-associated proteins, FtsA and ZipA. Although interaction between an FtsZ monomer and either of these proteins is of low affinity, high affinity is achieved through avidity - polymerization linked CCTPs interacting with the membrane tethers. The placement of the Z ring at midcell is ensured by antagonists of FtsZ polymerization that are positioned within the cell and target FtsZ filaments through the CCTP. The placement of the ring is reinforced by a protein network that extends from the terminus (Ter) region of the chromosome to the Z ring. Once the Z ring is established, additional proteins are recruited through interaction with FtsA, to form the divisome. The assembled divisome is then activated by FtsN to carry out septal peptidoglycan synthesis, with a dynamic Z ring serving as a guide for septum formation. As the septum forms, the cell wall is split by spatially regulated hydrolases and the outer membrane invaginates in step with the aid of a transenvelope complex to yield progeny cells.

FtsZ-ring Architecture and Its Control by MinCD.

In bacteria and archaea, the most widespread cell division system is based on the tubulin homologue FtsZ protein, whose filaments form the cytokinetic Z-ring. FtsZ filaments are tethered to the membrane by anchors such as FtsA and SepF and are regulated by accessory proteins. One such set of proteins is responsible for Z-ring's spatiotemporal regulation, essential for the production of two equal-sized daughter cells. Here, we describe how our still partial understanding of the FtsZ-based cell division process has been progressed by visualising near-atomic structures of Z-rings and complexes that control Z-ring positioning in cells, most notably the MinCDE and Noc systems that act by negatively regulating FtsZ filaments. We summarise available data and how they inform mechanistic models for the cell division process.

Bacterial Actins.

A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

WhmD promotes the assembly of Mycobacterium smegmatis FtsZ: A possible role of WhmD in bacterial cell division

WhmD is considered to have a role in the septation and division of Mycobacterium smegmatis cells. Since FtsZ is the central protein of the septum, we determined the effect of WhmD on the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ) in vitro. WhmD increased both the rate and extent of the assembly of MsFtsZ in vitro. WhmD also increased the amount of polymerized MsFtsZ as evident from a sedimentation assay. Further, the assembly promoting activity of WhmD occurred in the presence of GTP. MsFtsZ polymerized to form thin filaments in the absence of WhmD while MsFtsZ formed thick filaments in the presence of WhmD suggesting that WhmD enhanced the bundling of MsFtsZ filaments. Interestingly, WhmD neither suppressed the dilution-induced disassembly of FtsZ filaments nor significantly altered the GTPase activity of FtsZ. Using size exclusion chromatography, circular dichroism and fluorescence spectroscopy, WhmD was found to bind to MsFtsZ in vitro. The results showed that WhmD can promote the assembly of FtsZ and indicated that WhmD may play a role in the division of M. smegmatis cells by assisting the polymerization of FtsZ.

Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein.

The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function.

Inside Cover: Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets (Angew. Chem. Int. Ed. 43/2016)

Self-organizing patterns of purified Min proteins, which are spatial regulators of bacterial cell division, and the cell division protein FtsZ were observed when the proteins were reconstituted in emulsion droplets, as reported by P. Schwille and co-workers in their Communication on page 13455 ff. The cover illustrates FtsZ filament bundles (blue) and Min proteins (red) in the lumen of a droplet, with a lipid monolayer (green) at the droplet interface (image by G. Chwastek).

FtsZ-Dependent Elongation of a Coccoid Bacterium.

ABSTRACTA mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZG193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZG193D filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci.

Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers.

Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments in the presence of GTP. Since the discovery of the Z-ring 25years ago, various models for the role of FtsZ have been suggested. However, important information about the architecture and dynamics of FtsZ filaments during cytokinesis is still missing. One reason for this lack of knowledge has been the small size of bacteria, which has made it difficult to resolve the orientation and dynamics of individual FtsZ filaments in the Z-ring. While superresolution microscopy experiments have helped to gain more information about the organization of the Z-ring in the dividing cell, they were not yet able to elucidate a mechanism of how FtsZ filaments reorganize during assembly and disassembly of the Z-ring. In this chapter, we explain how to use an in vitro reconstitution approach to investigate the self-organization of FtsZ filaments recruited to a biomimetic lipid bilayer by its membrane anchor FtsA. We show how to perform single-molecule experiments to study the behavior of individual FtsZ monomers during the constant reorganization of the FtsZ-FtsA filament network. We describe how to analyze the dynamics of single molecules and explain why this information can help to shed light onto possible mechanism of Z-ring constriction. We believe that similar experimental approaches will be useful to study the mechanism of membrane-based polymerization of other cytoskeletal systems, not only from prokaryotic but also eukaryotic origin.

Collective effects of torsion in FtsZ filaments.

Recent evidence points to the presence of torsion in FtsZ bonds. In addition, experiments with FtsZ mutants on surfaces resulted in new aggregates that cannot be explained by older models for FtsZ dynamics. We use an interaction model for FtsZ derived from molecular dynamics simulations and expand a fine-grained lattice model used to describe FtsZ aggregates on a surface. This new model includes different anchoring angles for the monomers and allows bond twist, two ingredients that oppose each other resulting in a more dynamic and interesting system. We study the role and importance of these conflicting elements and how the aggregates are characterized by the different interaction parameters.

Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling.

Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

A Microfluidic Approach to the Study of Z-Ring Dynamics Formation in Liposomes

Thanks to recent improvements in biological engineering, the idea of building a synthetic minimal cell that is able to replicate itself using a minimal amount of components has recently emerged as an ambitious goal that may be feasible to achieve in the long term. Following a bottom-up approach, two essential elements are required to provide the fundamental functionality of cellular division: a lipid membrane for confinement and a protein machinery to carry out fission. We have developed a technique to produce cell-size liposomes by means of a microfluidic device, which provides an innovative way to obtain monodisperse membrane confinements with a fine control over the concentrations of the solutions inside and outside the membrane (see the contribution by S. Deshpande in this conference). To these liposomes, we administer a minimal form of the divisome machinery of E. coli, viz., the proteins that constitute the Z-ring, a highly dynamic structure responsible for the membrane's constriction and fission. A central element of the Z-ring is FtsZ, a protein that, under GTP hydrolysis, polymerizes into filament structures that have been suggested to provide the force to constrict the membrane. Using our microfluidics and fluorescence microscopy, we temporally resolve the FtsZ filaments formation and their interaction with the membrane anchor proteins FtsA and ZipA, studying the entity of the liposome deformation to optimize the crowding conditions required for the divisome machinery to better perform its constrictive action on the membrane.

Towards the Reconstitution of Minimal Cell Division

What would be the minimal molecular machinery required to divide a cell? This obviously depends on many factors, such as the size and shape of the cell and the composition of its contents and boundaries. It also depends on the ability of this machinery to self-organize and self-assemble, and to overcome the thermodynamic and kinetic barriers for such a significant morphological transition. Inspired by the divisome of the E.coli bacterial cell, we presently aim to reconstitute the first steps towards the positioning and contraction of a ring-like filamentous structure in the middle of a model cell compartment. In our minimal system, the MinCDE proteins oscillate between the two compartment poles and thereby faithfully position cytoskeletal FtsZ filaments to the center zone of a rod-like membrane compartment. I will discuss how the spatial regulation of the different elements depend on molecular properties of the underlying proteins and the spatial features of the membrane compartment. Building on the insights gained from this model, perspectives to engineer a minimal modular system for cell geometry recognition and regulation will be given.