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In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

The MinCDE proteins help to select cell division sites in normal cylindrical Escherichia coli by oscillating along the long axis, preventing unwanted polar divisions. To determine how the Min system might function in cells with multiple potential division planes, we investigated its role in a round-cell rodA mutant. Round cells lacking MinCDE were viable, but growth, morphology and positioning of cell division sites were abnormal relative to Min+ cells. In round cells with a long axis, such as those undergoing cell division, green fluorescent protein (GFP) fusions to MinD almost always oscillated parallel to the long axis. However, perfect spheres or irregularly shaped cells exhibited MinD movement to and from multiple sites on the cell surface. A MinE-GFP fusion exhibited similar behavior. These results indicate that the Min proteins can potentially localize anywhere in the cell but tend to move a certain maximum distance from their previous assembly site, thus favoring movement along the cell's long axis. A new model for the spatial control of division planes by the Min system in round cells is proposed.

The site of cell division in Escherichia coli is defined by formation of the Z ring between the two segregated daughter nucleoids. Positioning of the Z ring, composed of the highly conserved and tubulin-like FtsZ protein, appears to be negatively regulated by both the nucleoid and the oscillating MinCD inhibitor proteins. MukB protein is probably involved in nucleoid condensation, and in the absence of MukB, the negative effect of the nucleoid on Z rings appears to be partially suppressed. In this study, we examined the localization of Z rings in cells lacking both the Min system and MukB. In the Deltamin DeltamukB double null mutant, essentially all nucleoid-free zones, either at the cell poles or at non-polar sites between nucleoids, contained Z rings. However, a significant proportion of Z rings also formed on top of nucleoids. Interestingly, Z ring clusters often formed at gaps between nucleoids, and some of the rings within the clusters were clearly positioned on top of nucleoids. These results provide further evidence that the negative topological effect of nucleoids in cells lacking MukB is partially but not totally suppressed, and that the absence of the Min system allows more promiscuous Z ring formation.

To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C. Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.

The Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ.

To understand further the role of the nucleoid and the min system in selection of the cell division site, we examined FtsZ localization in Escherichia coli cells lacking MinCDE and in parC mutants defective in chromosome segregation. More than one FtsZ ring was sometimes found in the gaps between nucleoids in min mutant filaments. These multiple FtsZ rings were more apparent in longer cells; double or triple rings were often found in the nucleoid-free gaps in ftsI min and ftsA min double mutant filaments. Introducing a parC mutation into the ftsA min double mutant allowed the nucleoid-free gaps to become significantly longer. These gaps often contained dramatic clusters of FtsZ rings. In contrast, filaments of the ftsA parC double mutant, which contained active MinCDE, assembled only one or two rings in most of the large nucleoid-free gaps. These results suggest that all positions along the cell length are competent for FtsZ ring assembly, not just sites at mid-cell or at the poles. Consistent with previous results, unsegregated nucleoids also correlated with a lack of FtsZ localization. A model is proposed in which both the inhibitory effect of the nucleoid and the regulation by MinCDE ensure that cells divide precisely at the midpoint.

The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli, we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.

To gain further insight into the structural relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell division protein, we tested the effect of tubulin assembly inhibitors on FtsZ assembly. Common tubulin inhibitors, such as colchicine, colcemid, benomyl, and vinblastine, had no effect on Ca2+-promoted GTP-dependent assembly of FtsZ into polymers. However, the hydrophobic probe 5, 5'-bis-(8-anilino-1-naphthalenesulfonate) (bis-ANS) inhibited FtsZ assembly. The potential mechanisms for inhibition are discussed. Titrations of FtsZ with bis-ANS indicated that FtsZ has one high affinity binding site and multiple low affinity binding sites. ANS (8-anilino-1-naphthalenesulfonate), a hydrophobic probe similar to bis-ANS, had no inhibitory effect on FtsZ assembly. Because tubulin assembly has also been shown to be inhibited by bis-ANS but not by ANS, it supports the idea that FtsZ and tubulin share similar conformational properties. Ca2+, which promotes GTP-dependent FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesting that Ca2+ binding induces changes in the hydrophobic conformation of the protein. Interestingly, depletion of bound Ca2+ with EGTA further enhanced bis-ANS fluorescence. These findings suggest that both binding and dissociation of Ca2+ are capable of inducing FtsZ conformational changes, and these changes could promote the GTP-dependent assembly of FtsZ.

Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. coli cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and ftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in ftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.

FtsZ, a tubulin-like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis. It is not known what triggers FtsZ ring assembly. In this work, we use a FtsZ-green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy. We show that FtsZ polymers can assemble dynamically in solution in a GTP-dependent manner. Initially, FtsZ nucleation centers grow into aster-like structures that dramatically resemble microtubule organizing centers. As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo. Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly. FtsZ can undergo repeated GTP-dependent assembly and disassembly in solution by sequential addition and removal of Ca2+. In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration. Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell.