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PubMed1999

Attachment of the yeast Rhodosporidium toruloides is mediated by adhesives localized at sites of bud cell development.

The basidiomycetous yeast Rhodosporidium toruloides (anamorph, Rhodotorula glutinis) is a common phylloplane epiphyte with biocontrol potential. To understand how R. toruloides adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att-) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type R. toruloides was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att- mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of R. toruloides to both polystyrene and barley leaf surfaces.

Journal Article↗
PubMed1996

Effects of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridomas.

Membrane lipid organization and membrane fluidity affect cell functions. The effects of supplementing culture media with a lipid mixture (Ex-Cyte) containing cholesterol, phospholipids and fatty acids on cell growth, antibody production and membrane composition and dynamics in two hybridoma cell lines were studied. A49 cells decreased immunoglobulin production but cell growth increased. Lipids had no effect on the cell growth rate of B9 cells but increased immunoglobulin production and productivity. The fluidity of the deep areas of the plasma membrane and cytoplasmic organelles increased in the two cell lines. There was increased fluidity of the polar regions of the plasma membrane and a decreased phosphatidylethanolamine/phosphatidylcholine ratio in A49 cells. B9 cells underwent no change in fluidity of the polar regions but the phosphatidylinositol content was increased, together with higher monoclonal antibody production. These results demonstrate that antibody production is not linked to the dynamic properties of the membrane, even though changes in the membrane phosphatidylinositol content are associated with the final step of antibody secretion, but that the action of phospholipids and fatty acids on cell growth is membrane-associated.

Animals↗
PubMed2002

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude-Nernst equation.

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1-10 mus time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (-26 A(3)) and reaction enthalpy (-0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, -1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of approximately 4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe(4)S(4))(Cys(4)) with charge change from -1 to -2. The small volume change in PS II is explained by sub-mus electron transfer from Y(Z) anion to P(680) cation, in which charge is only moved from the Y(Z) anion to the Q(A) with no charge separation or with rapid proton transfer from oxidized Y(Z) to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P(680) cation.

Journal Article↗
PubMed1966

High resolution autoradiography of Escherichia coli cells infected with bacteriophage R17.

Granboulan, Nicole (Institute de Recherches sur le Cancer, Villejuif, Seine, France), and Richard M. Franklin. High-resolution autoradiography of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:849-857. 1966.-The ultrastructural alterations in Escherichia coli infected with the RNA bacteriophage R17 were further investigated by means of the technique of high-resolution autoradiography. Tritiated precursors to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein were employed in separate experiments. A striking inhibition of cellular RNA, DNA, and protein synthesis was noted. Whereas normal RNA synthesis occurs in the nucleoid, in infected cells RNA synthesis is predominantly cytoplasmic, but later in the latent period, and during the stage of active viral growth, the label is localized in a polar region. In the late stages of viral growth, RNA synthesis occurs only around the crystals. Protein synthesis also becomes localized in a polar region, but DNA synthesis remains confined to the nucleoid. Under conditions of chloramphenicol inhibition of viral-coat protein synthesis, RNA label is localized in the paranuclear lesion, providing further indication that RNA forms this fibrillar structure.

Amino Acids↗
PubMed1994

Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud.

Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 micrograms/ml) and physiological (beads soaked in 10 micrograms/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmacologic and physiological doses RA on limb bud tissue.

Animals↗
PubMed1986

Alterations in muscle spindle morphology in advanced stages of murine muscular dystrophy.

Muscle spindles in the soleus of 1-year-old dystrophic mice of the C57BL/6J dy2J/dy2J strain were studied by microscopic and morphometric methods, and comparisons were made with those in age-matched normal tissue. Transverse epon sections were cut through various regions of an individual receptor, and subsequent 90 degrees reorientation enabled longitudinal examination of the same spindle. In dystrophy, alterations were detected in the outer capsule and consisted of a significant increase in its overall thickness in equatorial regions. Perineurial proliferation accompanied histiocyte and collagen infiltration. Within the equator, intrafusal fibers and sensory terminals appeared unaffected by dystrophy. Alterations in the intrafusal fibers were restricted to polar zones where the mean diameters of chain and bag fibers were significantly reduced. Polar chain fibers exhibited a greater degree of atrophy in dystrophy with a 40% diminution in size. Ultrastructural changes in intrafusal fiber polar regions were less pronounced compared to the surrounding dystrophic muscle. Mitochondrial alterations in affected intrafusal fibers included intramatrix inclusions and glycogen deposition. Vacuolization of the sarcoplasmic reticulum and subsarcolemmal tubular aggregates were also observed in polar regions of dystrophic chain fibers. Regional variation in spindle involvement in advanced murine dystrophy provides evidence that the equatorial contents of this receptor are sequestered from the deleterious effects of the disease. Capsular thickening in the equator may be an adaptive response, preventing the intrafusal fibers from undergoing the moderate change and atrophy observed at their polar ends.

Animals↗
PubMed1999

Interactions between growth factors and gap junctional communication in developing systems.

In the vertebrate limb bud fibroblast growth factor (FGF) 4 secreted by cells of the posterior apical ectodermal ridge controls digit pattern, which is directed by polarizing cells in the posterior mesenchyme at the tip of the bud. FGF4 also controls the expression of gap junctions in the limb. Both chick and mouse limb bud mesenchyme express connexin 32 (Cx32; beta 1) and Cx43 (alpha 1), although not in the same gap junction plaques. Quantitative analysis reveals two gradients of gap junctions: from posterior to anterior in the subapical mesenchyme and from distal to proximal along the bud. The highest gap junction density is associated with the polarizing region. Micromass cultures of chick and mouse posterior and anterior mesenchyme cells were used to assess the ability of FGF4 to modulate gap junctional communication. Posterior mesenchyme (polarizing region) cells express a population of gap junctions that are highly sensitive to FGF4, whereas gap junctions between anterior mesenchyme cells are completely insensitive to FGF4. FGF4 doubles gap junction density, intercellular communication and the polarizing capacity of posterior mesenchyme cells, restoring polarizing capacity to in vivo levels. We conclude that gap junctional communication and polarizing capacity are intimately linked. Interactions between signalling molecules and junctional communication may play an important role in controlling development.

Amphibians↗
PubMed2001

On site bioremediation of hydrocarbon-contaminated Arctic tundra soils in inoculated biopiles.

There is a need to develop technology to allow the remediation of soil in polar regions that have been contaminated by hydrocarbon fuel spills. Bioremediation is potentially useful for this purpose, but has not been well demonstrated in polar regions. We investigated biopiles for on-site bioremediation of soil contaminated with Arctic diesel fuel in two independent small-scale field experiments at different sites on the Arctic tundra. The results were highly consistent with one another. In biopiles at both sites, extensive hydrocarbon removal occurred after one summer. After 1 year in treatments with optimal conditions, total petroleum hydrocarbons were reduced from 196 to below 10 mg per kg of soil at one site, and from 2,109 to 195 mg per kg of soil at the other site. Addition of ammonium chloride and sodium phosphate greatly stimulated hydrocarbon removal and indicates that biodegradation was the primary mechanism by which this was achieved. Inoculation with cold-adapted, mixed microbial cultures further stimulated hydrocarbon removal during the summer immediately following inoculation. At one site, soil temperature was monitored during the summer season, and a clear plastic cover increased biopile soil temperature, measured as degree-day accumulation, by 30-49%. Our results show that on-site bioremediation of fuel-contaminated soil at Arctic tundra sites is feasible.

Arctic Regions↗
PubMed2002

Stabilities and conformations of Alzheimer's beta -amyloid peptide oligomers (Abeta 16-22, Abeta 16-35, and Abeta 10-35): Sequence effects.

Previously, we have studied the minimal oligomer size of an aggregate amyloid seed and the mechanism of seed growth with a multilayer beta-sheet model. Under high temperature simulation conditions, our approach can test the stability of possible amyloid forms. Here, we report our study of oligomers of Alzheimer's amyloid beta-peptide (Abeta) fragments 16-22, 16-35, and 10-35 (abbreviated Abeta(16-22), Abeta(16-35), and Abeta(10-35), respectively). Our simulations indicate that an antiparallel beta-sheet orientation is the most stable for the Abeta(16-22), in agreement with a solid state NMR-based model [Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W., et al. (2000) Biochemistry 39, 13748-13759]. A model with twenty-four Abeta(16-22) strands indicates a highly twisted fibril. Whereas the short Abeta(16-22) and Abeta(24-36) may exist in fully extended form, the linear parallel beta-sheets for Abeta(16-35) appear impossible, mainly because of the polar region in the middle of the 16-35 sequence. However, a bent double-layered hairpin-like structure (called hook) with the polar region at the turn forms parallel beta-sheets with higher stability. An intra-strand salt-bridge (D23-K28) stabilizes the bent hairpin-like hook structure. The bent double-beta-sheet model for the Abeta(10-35) similarly offers oligomer stability.

Alzheimer Disease↗
PubMed2004

Photophysical properties of 2-amino-9,10-anthraquinone: evidence for structural changes in the molecule with solvent polarity.

The photophysical properties of 2-amino-9,10-anthraquinone (2AAQ) have been investigated in different solvents and solvent mixtures and correlated with the Lippert-Mataga solvent polarity parameter, Deltaf. In the low solvent polarity region with Deltaf < ca. 0.1, the dye shows unusually high fluorescence quantum yields (Phif) and lifetimes (tauf) in comparison to those in other solvents of medium to high polarities. Similarly, the radiative rate constants (kf) are relatively lower and the non-radiative rate constants (knr) are relatively higher in the low polarity solvents in comparison to those in the medium to high polarity solvents. The current results have been rationalized assuming that the dye adopts different structural forms below and above the Deltaf value of approximately 0.1. It is inferred that in the low solvent polarity region the dye exists in a non-planar structure, with its 2-NH2 plane away from that of the 9,10-anthraquinone moiety in the ground state. In solvents of medium to high polarities, the dye exists in a polar intramolecular charge transfer (ICT) structure, where the amino lone pair of the 2-NH2 group is in strong resonance with the anthraquinone pi-cloud in the ground state. In all the solvents, however the dye is inferred to exist in the ICT structure in its excited (S1) state. Supportive evidence for the above hypothesis has been obtained from the solvent polarity effect on the Stokes' shifts for the dye. Quantum chemical studies on the structures of 2AAQ dye in the gas phase also give qualitative support for the inferences drawn from the photophysical properties of the dye in different solvents.

Anthraquinones↗
PubMed2006

Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Muller glia cells.

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Müller glia (MG) cells. We demonstrate that the Crb1-/- phenotype, as shown in Crb1-/- mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1-/- primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. We conclude that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells.

Animals↗
PubMed1995

Vertebrate limb development.

The recent identification of Wnt-7a as a signalling molecule in dorsal/ventral patterning means that we now have a known signal for control of each of the three limb axes. Fibroblast growth factors can allow proximal/distal patterning and Sonic hedgehog gene expression signals anterior/posterior patterning. Networks of these signals not only coordinate cell responses, but also mutually maintain each other. A positive feedback loop is established which coordinates expression of Sonic hedgehog in mesenchyme cells of the polarizing region and Fgf-4 expression in overlying apical ridge ectoderm. Wnt-7a expression in dorsal ectoderm also influences Sonic hedgehog expression in the polarizing region. Initiation of development of a complete limb can be achieved with just one signal, a growth factor.

Animals↗
PubMed2005

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan.

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.

2-Naphthylamine↗
PubMed1981

The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells.

When metaphase PtK1 cells are cooled to 6-8 degrees C for 4-6 h the free, polar, and astral spindle microtubules (MTs) disassemble while the MTs of each kinetochore fiber cluster together and persist as bundles of cold-stable MTs. These cold-stable kinetochore fibers are similar to untreated kinetochore fibers in both their length (i.e., 5-6 micrometer) and in the number of kinetochore-associated MTs (i.e., 20-45) of which they are comprised. Quantitative information concerning the lengths of MTs within these fibers was obtained by tracking individual MTs between serial transverse sections. Approximately 1/2 of the kinetochore MTs in each fiber were found to run uninterrupted into the polar region of the spindle. It can be inferred from this and other data that a substantial number of MTs run uninterrupted between the kinetochore and the polar region in untreated metaphase PtK1 cells.

Animals↗
PubMed1977

Mitochondrial heredity of resistance to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of cytochrome b oxidation, in Saccharomyces cerevisiae.

3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), an inhibitor of cytochrome b oxidation, has been used for the selection of three resistant mutants (diur) of Saccharomyces cerevisiae. The mutant diur-64 exhibits in vivo cross-resistance to antimycin A while diur-34 and diur-1 are more sensitive to antimycin A than the parental strain. The three mutants exhibit mitochondrial inheritance according to the following criteria: mitotic segregation of diuron-resistant and diuron-sensitive diploids is obtained among the diploid progeny of a cross between diur and dius; non-Mendelian segregation of diuron resistance (4:0) is observed in spores of tetrads issued from diuron-resistant diploid; extensive ethidium bromide treatment leads to the formation of Q- mutants which no longer transmit diur and dius alleles. Evidence for two distinct diuron-resistant loci were obtained by allelism tests. Recombination analysis shows that diuron-resistance is not located in the polar region of the mitochondrial genome. The diur loci are not linked to the erythromycin locus since the upper limit in recombinants frequency (26%) for a non-polar region is obtained between diur and eryr. A low recombinants frequency (3%) is observed in crosses between diur-34 mutation and the two mutants cob1 and cob2 suggesting that diur-34 might be located between these two cytochrome-b-deficient loci. The resistance to diuron is also expressed in vitro since the oxidation rates of succinate by sonicated submitochondrial particles from the mutants are clearly less sensitive to diuron than that of the wild type.

Antimycin A↗
PubMed1987

Differential effects of combinations of phospholipase A2 and phospholipase C on the activity of rat epididymal nuclear and microsomal 4-ene steroid 5 alpha-reductase.

Epididymal 4-ene steroid 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal fractions, and the activity can be altered by modifying the phospholipids in the membrane environment. To investigate the membrane dependence of 4-ene steroid 5 alpha-reductase, we have treated nuclear and microsomal membranes with combinations of phospholipase A2 and phospholipase C, and examined the effects on 4-ene steroid 5 alpha-reductase activity. Sequential addition of phospholipase A2 and phospholipase C to the nuclear fraction, reduced the 4-ene steroid 5 alpha-reductase activity to approx 25% of the control level. Neither the nature of the phospholipase, nor the sequence of addition altered the inhibition. When both phospholipases were added simultaneously, nuclear 4-ene steroid 5 alpha-reductase activity was inhibited in a linear fashion, and in tests for cooperativity, the effects of phospholipase A2 and phospholipase C were clearly additive. The microsomal enzyme responded differently to sequential phospholipase treatments; if phospholipase A2 was followed by phospholipase C, or phospholipase C followed by phospholipase A2, the 4-ene steroid 5 alpha-reductase activity was, respectively, 13 and 27% of the control. In contrast, sequential addition of the same phospholipase reduced the activity of 4-ene steroid 5 alpha-reductase to approx 40% of the control level. Furthermore, simultaneous addition of phospholipase A2 and phospholipase C to the microsomal fraction, resulted in non-linearity of 4-ene steroid 5 alpha-reductase activity with time, whereas when added individually, linearity of 4-ene steroid 5 alpha-reductase was maintained. Consequently, it was not possible to test for cooperative effects of phospholipases on the microsomal 4-ene steroid 5 alpha-reductase. These findings suggest that for the nuclear 4-ene steroid 5 alpha-reductase, the polar and non-polar regions of the membrane environment have similar functions, which are most likely involved in the maintenance of the structural integrity of the enzyme. For the microsomal enzyme, the polar and non-polar regions of the membrane appear to have different functions, not only for the maintenance of enzyme integrity, but also in the mechanism at the active site.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1990

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells.

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.

Animals↗
PubMed2003

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site.

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.

Bacillus subtilis↗