Sodium dodecyl sulfate-sensitive septation in a mitomycin C-sensitive, mtc, mutant of Escherichia coli.

A mitomycin C-sensitive, mtc, mutant of Escherichia coli has an altered cell surface and is sensitive to sodium dodecyl sulfate (SDS). The mutant, M27, formed multinucleate nonseptated filaments in the presence of a low concentration of SDS (50 microgram/ml). When the culture grown at that concentration of SDS was diluted with an SDS-free medium, the filaments began to divide at a very rapid rate after a lag of about 20 min. Chloramphenicol inhibited this recovery division when added within 10 min after SDS dilution but did not inhibit the division when added 20 min after dilution. Penicillin G at a low concentration, which is enough to cause filamentation, had virtually no effect on the recovery division of SDS-induced filaments. The division of penicillin G-induced filaments was inhibited by SDS.     

Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli

We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA-negative cells. If SOS induction was blocked by lexA3 (Ind-), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.     

Cell growth and division in Escherichia coli: a common genetic control involved in cell division and minicell formation

Escherichia coli Div 124(ts) is a conditional-lethal cell division mutant formed from a cross between a mutant that produces polar anucleated minicells and a temperature-sensitive cell division mutant affected in a stage of cross-wall synthesis. Under permissive growth temperature (30 C), Div 124(ts) grows and produces normal progeny cells and anucleated minicells from its polar ends. When transferred to nonpermissive growth temperature (42 C), growth and macromolecular synthesis continue, but cell division and minicell formation are inhibited. Growth at 42 C results in formation of filamentous cells showing some constrictions along the length of the filaments. Return of the filaments from 42 to 30 C results in cell division and minicell formation in association with the constrictions and other areas along the length of the filaments. This gives rise to a "necklace-type" array of cells and minicells. Recovery of cell division is observed after a lag and is followed by a burst in cell division and finally by a return to the normal growth characteristic of 30 C cultures. Recovery of cell division takes place in the presence of chloramphenicol or nalidixic acid when these are added at the time of shift from 42 to 30 C, and indicates that a division potential for filament fragmentation is accumulated while the cells are at 42 C. This division potential is used for the production of both minicells and cells of normal length. The conditional-lethal temperature sensitive mutation controls a step(s) in cross-wall synthesis common to cell division and minicell formation.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera

In order to understand the mechanism of unequal division, polar body formation was investigated using the oocytes of the starfish, Asterina pectinifera. Cortical actin filaments were quantitatively measured after staining the maturing oocytes with fluorescently labeled phalloidin using a computer and image-processing software. Before polar body formation, at first the actin filaments at the animal pole decreased and subsequently the animal pole bulged. On the other hand, actin filaments surrounding the animal pole increased gradually and made a cleavage furrow around the animal pole as the bulge grew. Then the furrow ingressed and finally a polar body formed. When the surface force was calculated according to the cell shape, the surface force decreased at the animal pole but the force at the contractile ring increased. When by micromanipulation the mitotic apparatus was detached and translocated to the cortex other than the animal pole, polar body formation occurred all over the cortex of the oocyte, which indicates that the response of the whole cortex to the mitotic apparatus is equal. These results indicate that the decrease in the actin filaments and surface force near the centrosome of the mitotic apparatus as well as the increase in the actin filaments and surface force at some distance of the centrosome is important for cytokinesis.     

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradition delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.     

Structure and development of the egg of the glossiphoniid leech Theromyzon rude: reorganization of the fertilized egg during completion of the first meiotic division

Reorganization of the fertilized egg during completion of the first meiotic division was studied in the glossiphoniid leech Theromyzon rude. Rotation of the meiotic spindle, presumably as a result of changes in the length and arrangement of astral fibers, allows one of its poles to approach the prospective animal pole (AP), which appears as a differentiated region of the ectoplasm. The peripheral spindle pole is greatly modified during its anchorage to the AP and is dismantled upon emission of the first pole cell. Meanwhile, the central spindle pole is less modified and is reused during the second meiotic division. Redistribution of microvilli, as well as rearrangement of the ectoplasmic actin lattice, lead to remodeling of the egg surface. Emission of the first pole cell is preceded by a contraction wave that seems to arise by condensation of subcortical actin filaments at the equator of the egg. Poleward displacement of this wave causes evagination of the AP and ooplasmic segregation. A cytokinetic contractile ring forms by assembly of cortical actin filaments at the base of the AP evagination. When this process is disturbed by colchicine or cytochalasin B treatment, abortive or ghost pole cells may be formed.     

Quantitative analysis of cortical actin filaments during polar body formation in starfish oocytes

Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.     

Stalk Formation and Its Inhibition in Caulobacter crescentus

Estimates of average rates of stalk formation over several generations of growth in Caulobacter crescentus showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type C. crescentus grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

Fusome asymmetry and oocyte determination in Drosophila

Germline cysts containing 16 interconnected cells (cystocytes) are produced at an early stage of Drosophila oogenesis by progenitor cells known as cystoblasts that undergo four synchronous rounds of incomplete division. During cyst formation, a region of specialized, spectrin-rich cytoplasm called the fusome traverses the intercellular Connections (ring canals), linking individual cystocytes. Subsequently, 15 cystocytes begin to transport specific RNAs and other components into the remaining cell, the future oocyte. We used fusome-specific antibodies to characterize the early stages of cyst formation. During the first cystoblast division, a spherical mass of fusome material (the “spectrosome”) was associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associated with the pole of each mitotic spindle that remained in the mother cell, and only extended through the newly formed ring canals after each division was completed. These observations suggest that fusomes help establish a system of directional transport between cystocytes that underlies oocyte determination. © 1995 Wiley-Liss, Inc.     

Mutant of Escherichia coli with Thermosensitive Protein in the Process of Cellular Division

A new thermosensitive mutant of Escherichia coli deficient in cell division was isolated by means of membrane filtration after nitrosoguanidine mutagenesis. The mutant cells grow normally at 30 C but stop dividing immediately after shift to 42 C, resulting in multinucleated filaments lacking septa. The number of colony-forming units does not decrease for at least 6 hr at 42 C. The maximum length of the filaments is 10 to 16 times that of normal cells. Addition of a high concentration of NaCl fails to stimulate cell division at 42 C. The filaments formed at 42 C divide abruptly 30 min after shift to 30 C, and synchronous increase of cell number is shown for 3 hr. The macromolecular synthesis of protein and nucleic acids at 42 C is normal on the whole. The cell division shown after the shift from 42 to 30 C is observed in the absence of thymine, but not in the presence of chloramphenicol or in a medium deficient in amino acids. However, the filament can divide to some extent in the presence of chloramphenicol if some protein synthesis is allowed to proceed at 30 C before the addition of the antibiotic. The elongated cells divide at 42 C provided that they are exposed to 30 C before being shifted to high temperature.     

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.     

The effect of sublethal doses of rifampin on the sporulation of Clostridium botulinum.

Sublethal doses of rifampin (0-005 mug/ml), added to vegetatively growing cultures of a sporogenic mutant of Clostridium botulinum at inoculation time or after 4 h, resulted in a decrease of growth and in blockage of spore formation. But when rifampin was added 6 to 24 h after inoculation, normal growth and sporulation occurred, indicating that the time of addition was critical and that rifampin was most effective on rapidly dividing, exponential-phase cells. Ultrastructural studies showed that when rifampin was added at the time of inoculation, endospore development was blocked at stage III. During subsequent incubation (greater than 10 h) the cells lost their rigidity, and lysis of the mother cell was followed by that of the forespore. When the cultures were treated with rifampin at 4 h, about 40% of the cells were blocked at stage III and about 60% reached stages IV and V. Some showed excessive elongation and contained developing spores at each pole. They appeared to be derived from two daughter cells unable to form a division septum because of a specific inhibitory effect of rifampin on division. It would seem, therefore, that two daughter cells which are genetically coded to form endospores will do so irrespective of the development of a division septum, and the spores are formed at the 'old' polar regions.     

Localization of Surface Structures During Procaryotic Differentiation: Role of Cell Division in Caulobacter crescentus

Asymmetric cell division in Caulobacter crescentus produces two cell types, a stalked cell and a new swarmer cell, with characteristics surface structures. We have examined the role of the cell cycle in the differentiation of these two cells using the adsorption of bacteriophage øLC72, the assembly of the polar flagellum, and stalk formation as assays for changes in surface morphology. Previous studies of this aquatic bacterium [17, 25] have suggested that the replicating chromosome acts as a 'clock' in timing the formation of the flagellar filament at one pole of the new swarmer cell. The analysis of conditional cell cycle mutants presented here extends these results by showing that DNA synthesis is also required for adsorption of phage øLC72 and, more importantly, they also suggest that a late cell division step is involved in determining the spatial pattern in which the phage receptors and flagella are assembled. We propose that this cell division step is required for formation of 'organizational' centers which direct the assembly of surface structures at the new cell poles, and for the polarity reversal in assembly that accompanies swarmer cell to stalked cell development.     

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.     

The effect of centrosome UV microbeam irradiation on cell behavior. II. The radiation sequelae in the anaphase: the completion of division and the fate of the interphase cell]

Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.     

Actin filaments line up acrossTradescantia epidermal cells,anticipating wound-induced divison planes

Summary This paper describes the role of actin filaments in setting up the phragmosome — the transvacuolar device that anticipates the division plane — and in forming a supracellular system that seems to override cell boundaries. Tradescantia leaf epidermal cells were induced to divide by wounding the leaf. New division planes formed parallel to slits, and encircled puncture wounds — the new division planes lining up across cells, instead of the joints being off-set as in normal, unwounded tissue. Within 30 min after wounding, rhodamine phalloidin staining showed that a belt of fine, cortical actin filaments formed parallel to the wound. In the next stage, migration of nuclei to a wall adjacent to the wound, involved pronounced association of actin filaments with the nucleus. Migration could be inhibited with cytochalasin D, confirming the role of actin in traumatotaxis. Later still, actin strands were seen to line up from cell to cell, parallel to the wound, anticipating the future division plane. Next, actin filaments accumulated in this anticlinal plane, throughout the depth of the cell, thereby contributing to the formation of the phragmosome. The phragmosome has been shown in previous work (Flanders et al. 1990) to contain microtubules that bridge nucleus to cortex, and is now found to contain actin filaments. Actin filaments are therefore involved in the key stages of nuclear migration and division plane alignment. The supracellular basis of actin alignment is discussed.Dedicated to the memory of Professor Oswald Kiermayer     

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.