Role of the antiparallel double-stranded filament form of FtsA in activating the Escherichia coli divisome.

The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. Yet, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionallity of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

Insights into the assembly and regulation of the bacterial divisome.

The ability to split one cell into two is fundamental to all life, and many bacteria can accomplish this feat several times per hour with high accuracy. Most bacteria call on an ancient homologue of tubulin, called FtsZ, to localize and organize the cell division machinery, the divisome, into a ring-like structure at the cell midpoint. The divisome includes numerous other proteins, often including an actin homologue (FtsA), that interact with each other at the cytoplasmic membrane. Once assembled, the protein complexes that comprise the dynamic divisome coordinate membrane constriction with synthesis of a division septum, but only after overcoming checkpoints mediated by specialized protein-protein interactions. In this Review, we summarize the most recent evidence showing how the divisome proteins of Escherichia coli assemble at the cell midpoint, interact with each other and regulate activation of septum synthesis. We also briefly discuss the potential of divisome proteins as novel antibiotic targets.

Probing Membrane-Associated Cytoskeletal Oligomers of the Bacterial Divisome by Electron Microscopy and Tomography.

The cell division machinery or "divisome" of many bacteria, including Escherichia coli, contains homologs of tubulin (FtsZ) and actin (FtsA) that interact with each other to promote the synthesis of septal peptidoglycan. FtsA oligomers have an essential role as a track for tethering dynamically treadmilling FtsZ protofilaments to the cytoplasmic membrane. Other bacterial cytoskeletal oligomers such as MreB also assemble on and move along the membrane. Structures of these oligomers on membranes in vitro may mimic their behavior in the cell. Here, we describe a protocol to visualize FtsA oligomeric structures on membranes and their interactions with FtsZ protofilaments using negative stain transmission electron microscopy along with tomography.

Benzodioxane-Benzamides as FtsZ Inhibitors: Effects of Linker's Functionalization on Gram-Positive Antimicrobial Activity.

FtsZ is an essential bacterial protein abundantly studied as a novel and promising target for antimicrobials. FtsZ is highly conserved among bacteria and mycobacteria, and it is crucial for the correct outcome of the cell division process, as it is responsible for the division of the parent bacterial cell into two daughter cells. In recent years, the benzodioxane-benzamide class has emerged as very promising and capable of targeting both Gram-positive and Gram-negative FtsZs. In this study, we explored the effect of including a substituent on the ethylenic linker between the two main moieties on the antimicrobial activity and pharmacokinetic properties. This substitution, in turn, led to the generation of a second stereogenic center, with both erythro and threo isomers isolated, characterized, and evaluated. With this work, we discovered how the hydroxy group slightly affects the antimicrobial activity, while being an important anchor for the exploitation and development of prodrugs, probes, and further derivatives.

Benzodioxane-benzamides as promising inhibitors of Escherichia coli FtsZ.

The conserved process of cell division in bacteria has been a long-standing target for antimicrobials, although there are few examples of potent broad-spectrum compounds that inhibit this process. Most currently available compounds acting on division are directed towards the FtsZ protein, a self-assembling GTPase that is a central element of the division machinery in most bacteria. Benzodioxane-benzamides are promising candidates, but poorly explored in Gram-negatives. We have tested a number of these compounds on E. coli FtsZ and found that many of them significantly stabilized the polymers against disassembly and reduced the GTPase activity. Reconstitution in crowded cell-like conditions showed that FtsZ bundles were also susceptible to these compounds, including some compounds that were inactive on protofilaments in dilute conditions. They efficiently killed E. coli cells defective in the AcrAB efflux pump. The activity of the compounds on cell growth and division generally showed a good correlation with their effect in vitro, and our experiments are consistent with FtsZ being the target in vivo. Our results uncover the detrimental effects of benzodioxane-benzamides on permeable E. coli cells via its central division protein, implying that lead compounds may be found within this class for the development of antibiotics against Gram-negative bacteria.

Anchors: A way for FtsZ filaments to stay membrane bound.

Most bacteria use the tubulin homolog FtsZ to organize their cell division. FtsZ polymers initially assemble into mobile complexes that circle around a ring-like structure at the cell midpoint, followed by the recruitment of other proteins that will constrict the cytoplasmic membrane and synthesize septal peptidoglycan to divide the cell. Despite the need for FtsZ polymers to associate with the membrane, FtsZ lacks intrinsic membrane binding ability. Consequently, FtsZ polymers have evolved to interact with the membrane through adaptor proteins that both bind FtsZ and the membrane. Here, we discuss recent progress in understanding the functions of these FtsZ membrane tethers. Some, such as FtsA and SepF, are widely conserved and assemble into varied oligomeric structures bound to the membrane through an amphipathic helix. Other less-conserved proteins, such as EzrA and ZipA, have transmembrane domains, make extended structures, and seem to bind to FtsZ through two separate interactions. This review emphasizes that most FtsZs use multiple membrane tethers with overlapping functions, which not only attach FtsZ polymers to the membrane but also organize them in specific higher-order structures that can optimize cell division activity. We discuss gaps in our knowledge of these concepts and how future studies can address them.

Bacterial division ring stabilizing ZapA versus destabilizing SlmA modulate FtsZ switching between biomolecular condensates and polymers.

Cytokinesis is a fundamental process for bacterial survival and proliferation, involving the formation of a ring by filaments of the GTPase FtsZ, spatio-temporally regulated through the coordinated action of several factors. The mechanisms of this regulation remain largely unsolved, but the inhibition of FtsZ polymerization by the nucleoid occlusion factor SlmA and filament stabilization by the widely conserved cross-linking protein ZapA are known to play key roles. It was recently described that FtsZ, SlmA and its target DNA sequences (SlmA-binding sequence (SBS)) form phase-separated biomolecular condensates, a type of structure associated with cellular compartmentalization and resistance to stress. Using biochemical reconstitution and orthogonal biophysical approaches, we show that FtsZ-SlmA-SBS condensates captured ZapA in crowding conditions and when encapsulated inside cell-like microfluidics microdroplets. We found that, through non-competitive binding, the nucleotide-dependent FtsZ condensate/polymer interconversion was regulated by the ZapA/SlmA ratio. This suggests a highly concentration-responsive tuning of the interconversion that favours FtsZ polymer stabilization by ZapA under conditions mimicking intracellular crowding. These results highlight the importance of biomolecular condensates as concentration hubs for bacterial division factors, which can provide clues to their role in cell function and bacterial survival of stress conditions, such as those generated by antibiotic treatment.

Construction and Characterization of Functional FtsA Sandwich Fusions for Studies of FtsA Localization and Dynamics during Escherichia coli Cell Division.

FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsA's localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherrysw and FtsA-msfGFPsw acted as mild hypermorphs, as they were not toxic when overproduced, bypassed the essential cell division protein ZipA, and suppressed several thermosensitive fts alleles, although not as effectively as the prototypical hypermorph FtsA*. Overall, our results indicate that fluorescent FtsA sandwich fusions can be used as the sole FtsA in E. coli and thus should shed new light on FtsA dynamics during the cell division cycle in this model system. IMPORTANCE FtsA is a key conserved cell division protein, and E. coli is the most well studied model system for bacterial cell division. One obstacle to full understanding of this process is the lack of a fully functional fluorescent reporter for FtsA in vivo. Here, we describe a fluorescent fusion to E. coli FtsA that promotes efficient cell division in the absence of the native FtsA and can be used to monitor FtsA dynamics during cell division.

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

ZipA Uses a Two-Pronged FtsZ-Binding Mechanism Necessary for Cell Division.

In most bacteria, cell division is centrally organized by the FtsZ protein, which assembles into dynamic filaments at the division site along the cell membrane that interact with other key cell division proteins. In gammaproteobacteria such as Escherichia coli, FtsZ filaments are anchored to the cell membrane by two essential proteins, FtsA and ZipA. Canonically, this interaction was believed to be mediated solely by the FtsZ C-terminal peptide (CTP) domain that interacts with these and several other regulatory proteins. However, we now provide evidence of a second interaction between FtsZ and ZipA. Using site-specific photoactivated cross-linking, we identified a noncanonical FtsZ-binding site on ZipA on the opposite side from the FtsZ CTP-binding pocket. Cross-linking at this site was unaffected by the truncation of the FtsZ linker and CTP domains, indicating that this noncanonical site must interact directly with the globular core domain of FtsZ. Mutations introduced into either the canonical or noncanonical binding sites on ZipA disrupted photo-cross-linking with FtsZ and normal ZipA function in cell division, suggesting that both binding modes are important for normal cell growth and division. One mutation at the noncanonical face was also found to suppress defects of several other canonical and noncanonical site mutations in ZipA, suggesting there is some interdependence between the two sites. Taken together, these results suggest that ZipA employs a two-pronged FtsZ-binding mechanism. IMPORTANCE The tubulin homolog FtsZ plays a central early role in organizing bacterial cell division proteins at the cytoplasmic membrane. However, FtsZ does not directly interact with the membrane itself, instead relying on proteins such as FtsA to tether it to the membrane. In gammaproteobacteria, ZipA serves as a second essential membrane anchor along with FtsA. Although FtsA has a unique role in activating synthesis of the cell division septum, and ZipA may in turn activate FtsA, it was thought that both proteins interacted only with the conserved C terminus of FtsZ and were essentially interchangeable in their ability to tether FtsZ to the membrane. Here we challenge this view, providing evidence that ZipA directly contacts both the C terminus and the core domain of FtsZ. Such a two-pronged interaction between ZipA and FtsZ suggests that ZipA and FtsA may serve distinct membrane-anchoring roles for FtsZ.

Targeting the Achilles Heel of FtsZ: The Interdomain Cleft.

Widespread antimicrobial resistance among bacterial pathogens is a serious threat to public health. Thus, identification of new targets and development of new antibacterial agents are urgently needed. Although cell division is a major driver of bacterial colonization and pathogenesis, its targeting with antibacterial compounds is still in its infancy. FtsZ, a bacterial cytoskeletal homolog of eukaryotic tubulin, plays a highly conserved and foundational role in cell division and has been the primary focus of research on small molecule cell division inhibitors. FtsZ contains two drug-binding pockets: the GTP binding site situated at the interface between polymeric subunits, and the inter-domain cleft (IDC), located between the N-terminal and C-terminal segments of the core globular domain of FtsZ. The majority of anti-FtsZ molecules bind to the IDC. Compounds that bind instead to the GTP binding site are much less useful as potential antimicrobial therapeutics because they are often cytotoxic to mammalian cells, due to the high sequence similarity between the GTP binding sites of FtsZ and tubulin. Fortunately, the IDC has much less sequence and structural similarity with tubulin, making it a better potential target for drugs that are less toxic to humans. Over the last decade, a large number of natural and synthetic IDC inhibitors have been identified. Here we outline the molecular structure of IDC in detail and discuss how it has become a crucial target for broad spectrum and species-specific antibacterial agents. We also outline the drugs that bind to the IDC and their modes of action.

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics.

FtsZ is an essential and central protein for cell division in most bacteria. Because of its ability to organize into dynamic polymers at the cell membrane and recruit other protein partners to form a "divisome", FtsZ is a leading target in the quest for new antibacterial compounds. Strategies to potentially arrest the essential and tightly regulated cell division process include perturbing FtsZ's ability to interact with itself and other divisome proteins. Here, we discuss the available methodologies to screen for and characterize those interactions. In addition to assays that measure protein-ligand interactions in solution, we also discuss the use of minimal membrane systems and cell-like compartments to better approximate the native bacterial cell environment and hence provide a more accurate assessment of a candidate compound's potential in vivo effect. We particularly focus on ways to measure and inhibit under-explored interactions between FtsZ and partner proteins. Finally, we discuss recent evidence that FtsZ forms biomolecular condensates in vitro, and the potential implications of these assemblies in bacterial resistance to antibiotic treatment.

Computational Design and Development of Benzodioxane-Benzamides as Potent Inhibitors of FtsZ by Exploring the Hydrophobic Subpocket.

Multidrug resistant Staphylococcus aureus is a severe threat, responsible for most of the nosocomial infections globally. This resistant strain is associated with a 64% increase in death compared to the antibiotic-susceptible strain. The prokaryotic protein FtsZ and the cell division cycle have been validated as potential targets to exploit in the general battle against antibiotic resistance. Despite the discovery and development of several anti-FtsZ compounds, no FtsZ inhibitors are currently used in therapy. This work further develops benzodioxane-benzamide FtsZ inhibitors. We seek to find more potent compounds using computational studies, with encouraging predicted drug-like profiles. We report the synthesis and the characterization of novel promising derivatives that exhibit very low MICs towards both methicillin-susceptible and -resistant S. aureus, as well as another Gram positive species, Bacillus subtilis, while possessing good predicted physical-chemical properties in terms of solubility, permeability, and chemical and physical stability. In addition, we demonstrate by fluorescence microscopy that Z ring formation and FtsZ localization are strongly perturbed by our derivatives, thus validating the target.

Assembly of bacterial cell division protein FtsZ into dynamic biomolecular condensates.

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes, including cell division. Previous work revealed that FtsZ, a protein essential for cytokinesis in most bacteria, forms biomolecular condensates with SlmA, a protein that protects the chromosome from damage inflicted by the division machinery in Escherichia coli. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion by SlmA or to bacteria containing this protein. Here we report that FtsZ alone, under physiologically relevant conditions, can demix into condensates in bulk and when encapsulated in synthetic cell-like systems generated by microfluidics. Condensate assembly depends on FtsZ being in the GDP-bound state and on conditions mimicking the crowded environment of the cytoplasm that promote its oligomerization. Condensates are dynamic and reversibly convert into filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial division than initially envisioned.

Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment.

Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

The Nucleoid Occlusion Protein SlmA Binds to Lipid Membranes.

Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA's ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly.IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid membranes, adding a novel putative mechanism for regulating the local activity of these cell division proteins. We find that interaction between SlmA and lipid membranes is regulated by SlmA's DNA binding sites and protein binding partners as well as chemical conditions, suggesting that the SlmA-membrane interactions are important for fine-tuning the regulation of nucleoid integrity during cytokinesis.

Overproduction of a Dominant Mutant of the Conserved Era GTPase Inhibits Cell Division in Escherichia coli.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known in Escherichia coli that the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphic ftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling, ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring.IMPORTANCE All cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit in Escherichia coli, but its role in regulating E. coli cell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.

Bacterial Division: Journey to the Center of the Cell.

Most bacteria divide by corralling the tubulin-like FtsZ protein to mid-cell, where it assembles into a ring of treadmilling membrane-tethered oligomers. A study in this issue reveals new details about how FtsZ finds its way to the ring.