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.     

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Why on Earth: Self-assembly of the first bacterial cell to abundantand diverse bacterial species

About 80% of the evolutionary history of life on Earth is restricted to microorganisms which have had several billion years to speciate. The reasons for the origin (self-assembly) of life on Earth, bacterial cell division and why there are so many different bacteria and their global dispersal are discussed from an evolutionary perspective.     

A 4-aminofurazan derivative-A189-inhibits assembly of bacterial cell division protein FtsZ in vitro and in vivo

Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4-aminofurazan derivative was found to inhibit FtsZ GTPase with an IC(50) of 80 mug/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189-treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA-deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.     

Self‐organized partitioning of dynamically localized proteins in bacterial cell division

How cells manage to get equal distribution of their structures and molecules at cell division is a crucial issue in biology. In principle, a feedback mechanism could always ensure equality by measuring and correcting the distribution in the progeny. However, an elegant alternative could be a mechanism relying on self‐organization, with the interplay between system properties and cell geometry leading to the emergence of equal partitioning. The problem is exemplified by the bacterial Min system that defines the division site by oscillating from pole to pole. Unequal partitioning of Min proteins at division could negatively impact system performance and cell growth because of loss of Min oscillations and imprecise mid‐cell determination. In this study, we combine live cell and computational analyses to show that known properties of the Min system together with the gradual reduction of protein exchange through the constricting septum are sufficient to explain the observed highly precise spontaneous protein partitioning. Our findings reveal a novel and effective mechanism of protein partitioning in dividing cells and emphasize the importance of self‐organization in basic cellular processes.     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

Summary We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca²⁺ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells. Support for these studies was provided in part by the National Cancer Institute, Bethesda, Md., Contract N01-CP-01018.     

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage-T cell interactions

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl-OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257-264) presentation on K(b). Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257-264) peptide to K(b) on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1(-/-) mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl-OVA for OVA(257-264)/K(b) presentation, the binding of exogenously added OVA(257-264) to K(b) or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.     

Role of cell interactions in ascidian muscle and pigment cell specification

Summary Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca²⁺-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca²⁺-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.     

DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor

Summary Oligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations. This work was supported by the Deutsche Forschungsgemeinschaft (OB 66/2-1) and by the VW-Stiftung.     

GTP/GDP binding stabilizes bacterial cell division protein FtsZ against degradation by FtsH protease in vitro

Factors contributing to the stability of bacterial cell division protein FtsZ remain unknown. In order to identify FtsZ-stabilizing factor(s), we exploited FtsH protease-based in vitro FtsZ degradation assay system. Whole cell lysate from an ftsH-null strain of Escherichia coli inhibited degradation of FtsZ by FtsH in vitro. However, activated charcoal-treated lysate did not inhibit degradation. The loss of ability of the activated charcoal-treated lysate to inhibit degradation of FtsZ was restored when it was replenished with GTP, but not when replenished with other NTPs or dNTPs. The lysate did not protect either FtsZ deletion mutants, which do not bind GTP, or FtsH substrates, sigma(32) and cI-108 proteins, against FtsH. GDP and GTPgammaS also stabilized FtsZ against FtsH. Neither GTP nor GDP inhibited proteolytic activity of FtsH per se. These observations demonstrate that binding of GTP/GDP ligands is responsible for the proteolytic stability of FtsZ against FtsH.     

Control of Ehrlich cell division by zinc

The nutritional requirement for zinc in the proliferation of normal and malignant cells has been demonstrated in a number of animal studies. A distinction is made between the effect of zinc status upon the host during carcinogenesis and tumor growth. The present studies focus on the Ehrlich ascites tumor in mice fed a semipurified zinc-deficient diet along with defined concentration of zinc in the drinking water. This model of zinc deficiency is compared with others in which chelating agents are used to create zinc-deficient conditions or the microorganismEuglena gracilis is examined in a defined zinc-deficient medium. It is reported here that Ehrlich cells remain quiescent for several weeks in severely deficient mice, suggesting their restriction to a G₁ or G₀ state of the cell cycle. The kinetics of thymidine and uridine uptake and incorporation into DNA and RNA in Zn-normal and Zn-deficient tumors is consistent with the inhibition of thymidine kinase and DNA polymerase in the Zn-deprived system, but with little effect on RNA synthesis. The concentration of metabolites of these labeled nucleosides in Ehrlich cells is also consistent with a primary effect upon thymidine kinase. Although the ascites fluid Zn is depressed in Zn deficiency, total cellular zinc and its distribution among cell fractions is not significantly affected. It is suggested that these effects are specific in nature and not the result of a general lack of zinc for zinc metalloproteins and other binding sites in the cell.     

Genetic requirements for cell division in a genomically minimal cell

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Relationship between somatic cell count and bacterial pathogens in goat milk

The study aimed at evaluation of total count and pattern of somatic cells, as well as lactose content in relation to the type of bacterial pathogens in goat milk. The study was conducted on 66 Polish White Improved and Polish Fawn Improved dairy goats. A total of 487 milk samples were taken from day 30th, 60th and 200th of lactation for three years. The milk samples were divided into four groups: group 1 – containing no pathogens, group 2 – with minor pathogens up to 1000 CFU/mL such as coagulase-negative staphylococci (CNS), alpha-haemolytic streptococci, Enterococcus spp., Corynebacterium spp., group 3 – with minor pathogens (CNS) above 1 × 10³ CFU/mL of milk and group 4 – with major pathogens such as Streptococcus agalactiae, Staphylococcus intermedius and Staphylococcus aureus.In the majority of milk samples (64.9%) no pathogens were observed. The CNS were isolated from 25.3%, while the major pathogens were from 9.8% of milk samples. Both the major pathogens and high numbers of minor pathogens influenced the total somatic cell count (SCC) and lactose content. The percentage of leukocytes in the total somatic cells amounted to about 50% in the milk samples, which contained a high number of CNS or major pathogens. In the remaining samples this value reached only about 35%. Close relationship occurred between the presence of bacterial pathogens and total SCC, percentage of all leukocytes and their subpopulations in milk. The percentage of eosinophils and neutrophils in the total SCC were dramatically higher (p ≤ 0.0016) in samples of group 4 as compared to groups 1, 2 and 3. The percentage of monocytes was the highest in milk samples containing large numbers of minor pathogens. No relationship was found between the type of isolated bacterial pathogen and the percentage of lymphocytes in milk. In most samples, the presence of bacterial pathogens in goat milk led to the increase of the total SCC. However, the microbiological analysis showed that the bacterial pathogens were presented in about 20% of milk samples containing low SCC (below 1 × 10⁶/mL).     

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 μM or forskolin at 10 μM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 μM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 μM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists → changes in the level of calcium ions in cytoplasm → increased protein kinase activities → protein phosphorylation → modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.     

Characterization of testudine melanomacrophage linear,membrane extension processes—Cablepodia—By phase and atomic force microscopy

Summary Melanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates in their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.     

Tuning electron transfer by ester-group of chlorophylls in bacterial photosynthetic reaction center

Accessory chlorophylls (B(A/B)) in bacterial photosynthetic reaction center play a key role in charge-separation. Although light-exposed and dark-adapted bRC crystal structures are virtually identical, the calculated B(A) redox potentials for one-electron reduction differ. This can be traced back to different orientations of the B(A) ester-group. This tuning ability of chlorophyll redox potentials modulates the electron transfer from SP* to B(A).