Eukaryotic Tubulin Research Articles

Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration.

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis.

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and β-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

Bacterial Tubulins: A Eukaryotic-Like Microtubule Cytoskeleton

Ever since their discovery, bacterial tubulins, found in several Prosthecobacter species, have raised curiosity as they are closely related to eukaryotic tubulin. Deng and colleagues now present new evidence for the functional homology of the two cytoskeletal systems where in vitro reconstituted Btub-microtubules display eukaryote-like biochemical and dynamic properties.

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.

Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

Understanding the Asymmetric MipZ Gradient in Caulobacter Crescentus

Caulobacter crescentus is a freshwater α-proteobacterium that divides into two asymmetric daughter cells. These curved, rod-shaped cells differ in their chromosome replication fates and in their cell length at division. C. crescentus divides into a replication-competent ‘stalked’ cell and a smaller, replication-quiescent ‘swarmer’ cell. The site of cell division is dictated by the polymerization of the eukaryotic tubulin homologue, FtsZ. MipZ is an essential ATPase and inhibitor of FtsZ polymerization. MipZ forms an intracellular bipolar gradient that directs FtsZ to the lowest concentration of MipZ, which is found slightly towards the new pole as opposed to the exact mid-cell. This localization bias leads to two differently sized daughter cells. To better understand how MipZ directs FtsZ to this off-center location, we employed single-particle tracking photoactivated localization microscopy (sptPALM) to characterize the diffusive states and transition state kinetics of MipZ. We then used deterministic and stochastic simulations with these biophysical parameters to recreate the asymmetric MipZ gradient in silico. These simulations produce a distribution of cell lengths after cell division similar to that observed in vivo. These studies provide more insight into the asymmetric division of C. crescentus, but further questions remain regarding the nature of the asymmetric division.

The discovery of the prokaryotic cytoskeleton: 25th anniversary.

The year 2017 marks the 25th anniversary of the discovery of homologues of tubulin and actin in prokaryotes. Before 1992, it was largely accepted that tubulin and actin were unique to eukaryotes. Then three laboratories independently discovered that FtsZ, a protein already known as a key player in bacterial cytokinesis, had the “tubulin signature sequence” present in all α-, β-, and γ-tubulins. That same year, three candidates for bacterial actins were discovered in silico. X-ray crystal structures have since confirmed multiple bacterial proteins to be homologues of eukaryotic tubulin and actin. Tubulin and actin were apparently derived from bacterial precursors that had already evolved a wide range of cytoskeletal functions.

Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization

Chloroplast division is driven by a ring containing FtsZ1 and FtsZ2 proteins, which originated from bacterial FtsZ, a tubulin-like protein; however, mechanistic details of the chloroplast FtsZ ring remain unclear. Here, we report that FtsZ1 and FtsZ2 can heteropolymerize into a contractible ring ex vivo. Fluorescently labelled FtsZ1 and/or FtsZ2 formed single rings in cells of the yeast Pichia pastoris. Photobleaching experiments indicated that co-assembly of FtsZ1 and FtsZ2 imparts polarity to polymerization. Assembly of FtsZ chimaeras revealed that the protofilaments assemble via heteropolymerization of FtsZ2 and FtsZ1. Contraction of the ring was accompanied by an increase in the filament turnover rate. Our findings suggest that the evolutionary duplication of FtsZ in plants may have increased the mobility and kinetics of FtsZ ring dynamics in chloroplast division. Thus, the gene duplication and heteropolymerization of chloroplast FtsZs may represent convergent evolution with eukaryotic tubulin.

Comparison of nine reference genes for real-time quantitative PCR in roots and leaves during five developmental stages in carrot (Daucus carota L.)

ABSTRACTCarrot is a biennial plant with a nutritionally healthy composition and has long been recognised as an important model for genetic and molecular studies. The present study aimed to identify suitable reference genes for the normalisation of gene expression in carrot roots and leaves at different developmental stages. Three different software tools were applied to detect the stability of expression of nine candidate reference genes. The results showed that the best reference genes differed across tissues and developmental stages. Actin (ACTIN) was recommended as the most stable gene in carrot roots and leaves at all five developmental stages examined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be the least adequate gene, indicating that it should be avoided as a reference gene in carrot. Thus, we suggest ACTIN or different combinations of ACTIN, elongation factor 1α (EF-1α), eukaryotic translation initiation factor 4α (eIF-4α), tubulin β-7 (TUB), or polyubiquitin 10 (UBQ) for the normalisation of gene expression during carrot developmental processes. These results provide guidelines for future work on relative levels of gene expression in carrot using real-time reverse transcription-quantitative PCR (RT-qPCR).

Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.

Viriditoxin Induces G2/M Cell Cycle Arrest and Apoptosis in A549 Human Lung Cancer Cells

Viriditoxin is a fungal metabolite isolated from Paecilomyces variotii, which was derived from the giant jellyfish Nemopilema nomurai. Viriditoxin was reported to inhibit polymerization of FtsZ, which is a key protein for bacterial cell division and a structural homologue of eukaryotic tubulin. Both tubulin and FtsZ contain a GTPbinding domain, have GTPase activity, assemble into protofilaments, two-dimensional sheets, and protofilament rings, and share substantial structural identities. Accordingly, we hypothesized that viriditoxin may inhibit eukaryotic cell division by inhibiting tubulin polymerization as in the case of bacterial FtsZ inhibition. Docking simulation of viriditoxin to β-tubulin indicated that it binds to the paclitaxel-binding domain and makes hydrogen bonds with Thr276 and Gly370 in the same manner as paclitaxel. Viriditoxin suppressed growth of A549 human lung cancer cells, and inhibited cell division with G2/M cell cycle arrest, leading to apoptotic cell death.

3D-SIM Super-resolution of FtsZ and Its Membrane Tethers in Escherichia coli Cells

FtsZ, a bacterial homolog of eukaryotic tubulin, assembles into the Z ring required for cytokinesis. In Escherichia coli, FtsZ interacts directly with FtsA and ZipA, which tether the Z ring to the membrane. We used three-dimensional structured illumination microscopy to compare the localization patterns of FtsZ, FtsA, and ZipA at high resolution in Escherichia coli cells. We found that FtsZ localizes in patches within a ring structure, similar to the pattern observed in other species, and discovered that FtsA and ZipA mostly colocalize in similar patches. Finally, we observed similar punctate and short polymeric structures of FtsZ distributed throughout the cell after Z rings were disassembled, either as a consequence of normal cytokinesis or upon induction of an endogenous cell division inhibitor.

Six subgroups and extensive recent duplications characterize the evolution of the eukaryotic tubulin protein family.

Tubulins belong to the most abundant proteins in eukaryotes providing the backbone for many cellular substructures like the mitotic and meiotic spindles, the intracellular cytoskeletal network, and the axonemes of cilia and flagella. Homologs have even been reported for archaea and bacteria. However, a taxonomically broad and whole-genome-based analysis of the tubulin protein family has never been performed, and thus, the number of subfamilies, their taxonomic distribution, and the exact grouping of the supposed archaeal and bacterial homologs are unknown. Here, we present the analysis of 3,524 tubulins from 504 species. The tubulins formed six major subfamilies, α to ζ. Species of all major kingdoms of the eukaryotes encode members of these subfamilies implying that they must have already been present in the last common eukaryotic ancestor. The proposed archaeal homologs grouped together with the bacterial TubZ proteins as sister clade to the FtsZ proteins indicating that tubulins are unique to eukaryotes. Most species contained α- and/or β-tubulin gene duplicates resulting from recent branch- and species-specific duplication events. This shows that tubulins cannot be used for constructing species phylogenies without resolving their ortholog–paralog relationships. The many gene duplicates and also the independent loss of the δ-, ε-, or ζ-tubulins, which have been shown to be part of the triplet microtubules in basal bodies, suggest that tubulins can functionally substitute each other.

Identification of anti-tuberculosis agents that target the cell-division protein FtsZ.

Antibiotic resistance to Mycobacterium tuberculosis is a growing problem. Therefore, development of new anti-tuberculosis antibiotics is urgent for the control of tuberculosis (TB) infections. FtsZ, the homolog of eukaryotic tubulin, is a GTPase that assembles into cytokinetic Z rings essential for cell division in prokaryotic cells. FtsZ (filamentous temperature-sensitive protein Z) polymerizes in a GTP-dependent manner, and polymerization of FtsZ forms into dynamic protofilaments. In this study, we screened 20,000 compounds to identify inhibitors of GTPase activity of M. tuberculosis FtsZ. We found that 297F inhibited GTPase and polymerization of FtsZ, and reduced the amount of FtsZ polymers. Furthermore, 297F has anti-TB activity with low cytotoxicity and shows no antibacterial activities toward other Gram-positive or Gram-negative strains. In vitro, 297F also induced filamentation in Mycobacterium smegmatis. All results suggest that 297F inhibits bacterial proliferation by targeting M. tuberculosis FtsZ and it may be useful as a lead compound for developing anti-TB agents.

Characterization and in Vitro Inhibition Studies of Bacillus anthracis FtsZ: A Potential Antibacterial Target

FtsZ is an essential bacterial cell division protein that is an attractive target for the development of antibacterial agents. FtsZ is a homologue of eukaryotic tubulin, has GTPase activity, and forms a ring-type structure to initiate cell division. In this study, the FtsZ of Bacillus anthracis was cloned into a bacterial expression vector and overexpressed into Escherichia coli BL21 (DE3) cells. The overexpressed B. anthracis FtsZ was soluble and purified to homogeneity using Ni-His-tag affinity chromatography. Like other known FtsZs, the recombinant B. anthracis FtsZ also showed GTP-dependent polymerization, which was analyzed using both spectrophotometric and Transmission Electronic Microscopic (TEM) analysis. Using the purified FtsZ, we screened a naturally extracted chemical library to identify potent and novel inhibitors. The screening yielded three chemicals, SA-011, SA-059, and SA-069, that inhibited the in vitro polymerization activity of FtsZ in the micromolar range (IC50 of 55-168 μM). The inhibition potency was significantly comparable with that of berberine, a known potential inhibitor of FtsZ. Understanding the biochemical basis of the effect of these inhibitors on B. anthracis growth would provide a promising path for the development of new antianthracis drugs.

Variation in the Binding Pocket of an Inhibitor of the Bacterial Division Protein FtsZ Across Genotypes, Nucleotide States, and Species

The recent increase in antibiotic resistance in disease-causing bacteria calls for new approaches to drug target selection and drug development. Targeting proteins involved in the bacterial cell division process provides a novel mechanism of action that bypasses problems associated with the increasingly ineffective variants of older antibiotics. One potentially powerful target for inhibition is the bacterial cytoskeletal protein FtsZ, a GTPase homolog of eukaryotic tubulin. This highly conserved and essential protein acts as a scaffold to recruit other cell division machinery and can exert mechanical forces that may be responsible for constriction of the division site. Recent work on an allosteric inhibitor (PC190723) has shown promising in vitro and in vivo antimicrobial activities on Staphylococcus aureus, however the mechanism of drug action and its effect on FtsZ of other bacterial species is unclear. Here, we examine the structural environment of the FtsZ-PC190723 binding pocket using Molecular Dynamics simulations and PocketFEATURE, a statistical method that performs pairwise comparisons of small molecule binding sites based on 3D structure information. By comparing all currently existing FtsZ crystal structures to the crystalized FtsZ-PC190723 complex, we observe that species and nucleotide binding state have significant impact on the structural properties of the binding site, with a GDP-bound Staph-FtsZ displaying the highest similarity to the inhibitor complex. However, during extended Molecular Dynamics simulations, this similarity is negated upon introduction of point mutations known to nullify the inhibitor's antimicrobial affect. Together, these results give insight into the mechanism and specificity of PC190723 interaction with various bacterial FtsZ.

The FtsZ-Like Protein FtsZm of Magnetospirillum gryphiswaldense Likely Interacts with Its Generic Homolog and Is Required for Biomineralization under Nitrate Deprivation

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation. We analyzed the role of FtsZm in cell division and biomineralization together with the full-length MSR-1 FtsZ protein. Our results indicate that loss of FtsZm has a strong effect on microoxic magnetite biomineralization which, however, could be rescued by the presence of nitrate in the medium. Fluorescence microscopy revealed that FtsZm-mCherry does not colocalize with the magnetosome-related proteins MamC and MamK but is confined to asymmetric spots at midcell and at the cell pole, coinciding with the FtsZ protein position. In Escherichia coli, both FtsZ homologs form distinct structures but colocalize when coexpressed, suggesting an FtsZ-dependent recruitment of FtsZm. In vitro analyses indicate that FtsZm is able to interact with the FtsZ protein. Together, our data suggest that FtsZm shares key features with its full-length homolog but is involved in redox control for magnetite crystallization.

Phage ϕ29 protein p1 promotes replication by associating with the FtsZ ring of the divisome in Bacillus subtilis

During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage 29 protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperature-sensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage 29 DNA replication is impaired. Here we show that a functional fusion of protein p1 to YFP localizes at the medial region of Bacillus subtilis cells independently of other phage-encoded proteins. We also show that 29 protein p1 colocalizes with the B. subtilis cell division protein FtsZ and provide evidence that FtsZ and protein p1 are associated. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when 29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of 29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the 29 growth cycle.

A Super-Resolution Study of the Structural Organization of the E. Coli FTSZ-Ring by ZapA and ZapB

The first step in E. coli cell division is the localization of the FtsZ protein at midcell, where it polymerizes into a ring-like structure called the Z-ring. FtsZ is widely conserved throughout bacteria and is a structural homolog of eukaryotic tubulin. Understanding the organization of the Z-ring will provide insight into the force generation mechanism of bacterial cell division.In our previous work we used a super-resolution imaging method, photoactivated localization microscopy (PALM), to characterize the arrangement of FtsZ protofilaments inside the Z-ring1. We showed that the Z-ring is likely composed of a loose association of protofilaments overlapping in both the circumferential and radial direction of the Z-ring. Given that FtsZ protofilaments exhibit a low intrinsic self-interaction, how they are associated in vivo remains unknown.Recently a family of Z-ring associated proteins (Zap) have been shown to promote FtsZ polymerization in vitro and Z-ring formation in vivo. In this study, we investigated how ZapA and ZapB affect the organization of the Z-ring.We used live-cell PALM imaging to characterize the structure of the Z-ring in the absence of ZapA and ZapB. We found that in the absence of ZapA or ZapB, FtsZ adopts a variety of non-native structures characterized by a fractured appearance containing dispersed clusters of FtsZ. Similar FtsZ clusters have been observed to precede wild-type Z-ring assembly and are likely composed of multiple FtsZ protofilaments. Comparison of the FtsZ clusters observed in ΔzapA and ΔzapB to those observed in wild-type suggest that ZapA and ZapB may function to promote Z-ring assembly by corralling and consolidating higher-ordered FtsZ groupings.[1] G. Fu, T. Huang, J. Buss, C. Coltharp, Z. Hensel, J. Xiao, PLoS One 2010, 5, e12680.