Future Division Site Research Articles

Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

Stochastic assembly of chemoreceptor clusters in Escherichia coli

Chemoreceptors and cytoplasmic chemotaxis proteins in Escherichia coli form clusters that play a key role in signal processing. These clusters localize at cell poles and at specific positions along the cell body which correspond to future division sites, but the details of cluster formation and the mechanism of cluster distribution remain unclear. Here, we used fluorescence microscopy to investigate how the numbers and sizes of receptor clusters depend on the expression level of chemotaxis proteins and on the cell length. We show that the average cluster number saturates at high levels of protein expression at approximately 3.7 clusters per cell, well below the number of available positioning sites. Correspondingly, distances between clusters in filamentous cells saturate at an average of 1 mum but, even at saturating expression levels, individual cluster numbers and distances show a broad distribution around the mean. Our data imply a stochastic mode of cluster assembly, where a defined average interval between clusters along the cell body arises from competition between nucleation of new clusters and growth of existing clusters. Upon subsequent anchorage to defined lateral sites, clusters grow with rates that inversely depend on their size, and become polar upon several rounds of cell division.

Structural and mutational analysis of the cell division protein FtsQ

Bacterial cytokinesis requires the divisome, a complex of proteins that co-ordinates the invagination of the cytoplasmic membrane, inward growth of the peptidoglycan layer and the outer membrane. Assembly of the cell division proteins is tightly regulated and the order of appearance at the future division site is well organized. FtsQ is a highly conserved component of the divisome among bacteria that have a cell wall, where it plays a central role in the assembly of early and late cell division proteins. Here, we describe the crystal structure of the major, periplasmic domain of FtsQ from Escherichia coli and Yersinia enterocolitica. The crystal structure reveals two domains; the alpha-domain has a striking similarity to polypeptide transport-associated (POTRA) domains and the C-terminal beta-domain forms an extended beta-sheet overlaid by two, slightly curved alpha-helices. Mutagenesis experiments demonstrate that two functions of FtsQ, localization and recruitment, occur in two separate domains. Proteins that localize FtsQ need the second beta-strand of the POTRA domain and those that are recruited by FtsQ, like FtsL/FtsB, require the surface formed by the tip of the last alpha-helix and the two C-terminal beta-strands. Both domains act together to accomplish the role of FtsQ in linking upstream and downstream cell division proteins within the divisome.

Positioning of chemosensory clusters in E. coli and its relation to cell division

Chemotaxis receptors and associated signalling proteins in Escherichia coli form clusters that consist of thousands of molecules and are the largest native protein complexes described to date in bacteria. Clusters are located at the cell poles and laterally along the cell body, and play an important role in signal transduction. Much work has been done to study the structure and function of receptor clusters, but the significance of their positioning and the underlying mechanisms are not understood. Here, we used fluorescence imaging to study cluster distribution and follow cluster dynamics during cell growth. Our data show that lateral clusters localise to specific periodic positions along the cell body, which mark future division sites and are involved in the localisation of the replication machinery. The chemoreceptor cluster positioning is thus intricately related to the overall structure and division of an E. coli cell.

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

Decision of Spindle Poles and Division Plane by Double Preprophase Bands in a BY-2 Cell Line Expressing GFP–Tubulin

The preprophase band (PPB) of microtubules is thought to be involved in deciding the future division site. In this study, we investigated the effects of double PPBs on spindle formation and the directional decision of cytokinesis by using transgenic BY-2 cells expressing green fluorescent protein (GFP)-tubulin. At prophase, most of the cells with double PPBs formed multipolar spindles, whereas all cells with single PPBs formed normal bipolar spindles, clearly implicating the PPB in deciding the spindle poles. At metaphase, however, both cell types possessed the bipolar spindles, indicating the existence of correctional mechanism(s) at prometaphase. From prometaphase to metaphase, the spindles in double PPB cells altered their directions to become oblique to the cell-elongating axis, and these orientations were maintained in the phragmoplast and resulted in the oblique division planes. These oblique cell plates decreased when actin microfilaments were disrupted, and double actin-depleted zones (ADZs) appeared where the double PPBs had existed. These results suggest that the information necessary for proper cytokinesis may be transferred from the PPBs to the ADZs, even in the case of the double PPBs.

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast.

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.

The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae.

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

Assembly Dynamics of the Bacterial Cell Division Protein FtsZ: Poised at the Edge of Stability

FtsZ is a prokaryotic tubulin homolog that assembles into a ring at the future site of cell division. The resulting "Z ring" forms the framework for the division apparatus, and its assembly is regulated throughout the bacterial cell cycle. A highly dynamic structure, the Z ring exhibits continual subunit turnover and the ability to rapidly assemble, disassemble, and, under certain circumstances, relocalize. These in vivo properties are ultimately due to FtsZ's capacity for guanosine triphosphate (GTP)-dependent, reversible polymerization. FtsZ polymer stability appears to be fine-tuned such that subtle changes in its assembly kinetics result in large changes in the Z ring structure. Thus, regulatory proteins that modulate FtsZ's assembly dynamics can cause the ring to rapidly remodel in response to developmental and environmental cues.

The contractile ring

What is it? The contractile ring is a ring-shaped structure located just beneath the plasma membrane at the future division site in many, though not all, eukaryotic cell types. Composed of actin, myosin and many other proteins, it assembles in anaphase and contracts as cells divide. The contractile ring is responsible for cytokinesis in many eukaryotic cell types, and is thought to contribute to cell division by ‘squeezing’ the cell into two.How was it discovered? Theories of cytokinesis have been hotly debated for over a hundred years, ever since the first microscopists started observing dividing cells. Although a ‘muscle’-like band was proposed earlier, it wasn't until the late 1960s that Schroeder first observed filaments (of actin) at the cleavage furrow in sea urchin eggs by electron microscopy, and then showed that an anti-actin drug inhibits cytokinesis.Do all cells use this structure to divide? No. Although the contractile ring appears to be crucial for cytokinesis in many cell types, some cells do not have one or do not appear to need one. Although slime mold cells usually have a ring, myosin-deleted cells can still divide fairly normally on a plate even without a concentration of actin filaments at the cleavage furrow. Some animal cells, such as amphibian eggs, never have a complete ring, but form a spreading arc. Plants divide without one. How might cells divide without a ring? Depending on the cell type, membrane insertion through secretion also contributes to cytokinesis in varying degrees.How is the ring assembled? A set of conserved contractile ring proteins have been identified and characterized using genetic, biochemical and proteomic approaches in model organisms ranging from yeasts, plants, worms, and flies to sea urchin eggs and mammalian cells. These proteins, many of which organize actin in some manner, include: actin, myosin, septins, formins, Arp2/3 complex, tropomyosin, coronin, anillin, profilin, IQGAP, filiamin, MLCK, ROCK and so on. Actin filaments in the ring may be derived both from pre-formed filaments recycled from other actin structures and from new filaments newly polymerized by the ring's own actin nucleation centers. Myosin not only contributes force, but may also help bring the actin filaments together into a discrete ring. The small GTPase Rho is a key regulator of the process.How is the ring positioned? In animal cells, microtubules of the mitotic spindle somehow position the ring at a point equidistant from the two spindle poles. How this occurs is still highly controversial. In some cell types, overlapping astral microtubules from the two spindle poles that touch the cortex at the future division site may be the key spatial determinants, while in other cell types a bundle of microtubules known as the spindle midzone (or central spindle), may be responsible. Microtubules may regulate cleavage by sending out ‘signals’ that induce contractile ring formation, regulate contractility and direct membrane traffic. Other cell types use other spatial cues. For instance, fission yeast cells use the nucleus to position the cell division site, while budding yeast use cortical marks left over from previous cell divisions.How does the contractile ring divide the cell? A prevalent view is that that actin and myosin in the contractile ring exert squeezing forces leading to cleavage (at least in many cell types). Recent evidence shows that components of the ring are highly dynamic, suggesting that actin polymerization is also important for cleavage and may even contribute to force production. The ring also clearly has other roles, including organizing a membrane domain and targeting membrane insertion and trafficking.It seems that we don't know much about this pretty important universal process? Yes, cytokinesis is one of the frontiers of cell biology, filled with wildly different theories and controversies, studied in a large number of different model organisms in many different ways. Although certainly a universal process, it is becoming apparent that different cell types use similar but slightly different mechanisms. The advent of genomics, genetics, improved microscopy and proteomics promises rapid progress in sorting out the themes and variations in this fundamental process.Figure 1Contractile rings in a big and small cell. Sea urchin embryo (top) stained for actin filaments with rhodamine phalloidin and fission yeast cell (bottom) expressing a myosin light chain-GFP fusion protein. The fission yeast cell is approximately fourteen times smaller than the sea urchin cell.View Large Image | View Hi-Res Image | Download PowerPoint SlideWhere can I find out more?

Bacterial cell division spirals into control.

Directing proteins to specific subcellular addresses is a central problem in cell biology. Historically, perhaps because of their general lack of compartmentalized organelles, bacteria were viewed as relatively uniform at the subcellular level. However, with advances in fluorescence microscopy came the realization that bacteria, like their eukaryotic counterparts, segregate their proteins to different cellular regions (1). Recent studies have extended this principle by demonstrating that bacteria not only localize intracellular proteins to distinct regions of the cell, but that within these regions they can also organize proteins into discrete ordered structures. For example, the actin homologs MreB and Mbl form spirals along the long axis of Bacillus subtilis (2), and the cytokinetic tubulin homolog FtsZ localizes to a ring that goes through a spiral intermediate to reposition itself during B. subtilis sporulation (3). The work of Shih et al. (4) in this issue of PNAS demonstrates that the ability of intracellular proteins to organize into ordered structures is not exclusive to bacterial cytoskeletal elements. The authors make the exciting observation that proteins that were once thought to diffusely oscillate between the cell poles are in fact present in ordered spirals. Almost all cells divide at a highly reproducible location within the cell. This regularity requires the cell to first find its appropriate division site and then localize its division machinery to that site. This process is perhaps best characterized in the rod-shaped bacterium, Escherichia coli , where three proteins, MinC, MinD, and MinE, collaborate to position the cell division protein FtsZ at midcell (5, 6). FtsZ is a cytosolic tubulin homolog that can polymerize in vitro and assembles into a ring-like structure (the Z ring) in vivo (7, 8). The Z ring assembles at the middle of the cell, which is also the future division site, and together …

Golgi secretion is not required for marking the preprophase band site in cultured tobacco cells.

The preprophase band predicts the future cell division site. However, the mechanism of how a transient preprophase band fulfils this function is unknown. We have investigated the possibility that Golgi secretion might be involved in marking the preprophase band site. Observations on living BY-2 cells labeled for microtubules and Golgi stacks indicated an increased Golgi stack frequency at the preprophase band site. However, inhibition of Golgi secretion by brefeldin A during preprophase band formation did not prevent accurate phragmoplast fusion, and subsequent cell plate formation, at the preprophase band site. The results show that Golgi secretion does not mark the preprophase band site and thus does not play an active role in determination of the cell division site.

Multiple FtsZ Rings in a Pleomorphic Chloroplast in Embryonic Cap Cells of Pelargonium zonale.

The behaviors of the chloroplast FtsZ ring and outer cytosolic and inner stromal plastid dividing rings (PD rings) of chloroplast division were investigated in the higher plant Pelargonium zonale using immuno-fluorescence and electron microscopy. For these studies, we used embryonic cap cells from early embryos, in which small chloroplasts duplicate by multiple fission of a pleomorphic chloroplast. FtsZ proteins were localized to several rings at future division sites preceding contraction. FtsZ rings were found at the leading edges of the chloroplast constriction sites throughout division. In contrast, the PD rings were observed at the constriction sites after the formation of the FtsZ rings. These observations demonstrate that FtsZ rings differ from PD rings, and indicate that the FtsZ ring determines the dividing plane before the initiation of chloroplast contraction and then, in concert with the PD rings, promotes chloroplast division.

Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells.

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean alpha-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the "Golgi belt," accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.

Microtubule and actin-dependent movement of the formin cdc12p in fission yeast.

Although a number of gene products involved in cytokinesis have been identified, still little is known about how these proteins are localized to the proper site and assembled into a ring structure. How is the plane of cell division is positioned in the cell? Schizosaccharomyces pombe are simple rod-shaped eukaryotic cells that divide by medial fission using a medial contractile ring. S. pombe cdc12p encodes a member of the formin gene family, proteins with conserved roles in cytokinesis and actin organization. cdc12p is required specifically for the formation of the medial ring and is located in this ring during mitosis. Time-lapse microscopy of cells expressing GFP-cdc12p protein fusions reveals that during interphase, S. pombe cdc12p is present in a discrete, motile cytoplasmic particle that moves using both actin and microtubules. At the onset of mitosis, the spot moves to the future site of cell division and spreads out into a ring. These studies demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the site of contractile ring assembly. These findings suggest a potential mechanism for how the mitotic spindle positions the cell division plane in animal cells.

Compartmentalization of transcription and translation in Bacillus subtilis.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

Bacterial cell division.

Formation of the bacterial division septum is catalyzed by a number of essential proteins that assemble into a ring structure at the future division site. Assembly of proteins into the cytokinetic ring appears to occur in a hierarchial order that is initiated by the FtsZ protein, a structural and functional analog of eukaryotic tubulins. Placement of the division site at its correct location in Escherichia coli requires a division inhibitor (MinC), that is responsible for preventing septation at unwanted sites near the cell poles, and a topological specificity protein (MinE), that forms a ring at midcell and protects the midcell site from the division inhibitor. However, the mechanism responsible for identifying the position of the midcell site or the polar sites used for spore septum formation is still unclear. Regulation of the division process and its coordination with other cell cycle events, such as chromosome replication, are poorly understood. However, a protein has been identified in Caulobacter (CtrA) that regulates both the initiation of chromosome regulation and the transcription of ftsZ, and that may play an important role in the coordination process.

Movement of a cytokinesis factor cdc12p to the site of cell division

A key question in cytokinesis is how the plane of cell division is positioned within the cell. Although a number of cytokinesis factors involved in formation of the actomyosin contractile ring have been identified, little is known about how these factors are localized and assembled at the cell-division site. Cells of the fission yeast Schizosaccharomyces pombe divide using a medial actomyosin ring that assembles in early mitosis [1]. The S. pombe cdc12 gene encodes a formin, a member of a family of proteins that have functions in cytokinesis and cell polarity and that may bind Rho/Cdc42 GTPases, profilin and other actin-associated proteins [1–4]. The cdc12 protein (cdc12p) is required specifically for medial-ring assembly during cytokinesis and is a component of this ring [2,5]. In this study, cdc12p was found, during interphase, in a discrete, motile cytoplasmic spot that moved to the future site of cell division at the onset of mitosis. Three lines of evidence indicated that this cdc12p spot moved on both actin and microtubule networks: movement required either actin or microtubules; the spot was associated with actin and microtubule structures; and individual spots were seen to move along both microtubule and non-microtubule tracks. These findings demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the future site of cell division.

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell division

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs).During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The "actin-depleted zone" that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis.The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus.

Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites.

A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the beta-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.