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Biomedical subjects

C L Woldringh

Publications and source records attributed to C L Woldringh.

At least 37 records · Page 2Linked to original sources
PubMed1995

Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation.

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady-state generation time of 95-100 min, showing no change in cycle time during aging. The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 micro3 to a maximum of 34 micro3. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells. The 15-generation parent population, recovered from the chamber, had an average volume of 80 micro3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.

Cell Cycle↗
PubMed1994

Nucleoid partitioning in Escherichia coli during steady-state growth and upon recovery from chloramphenicol treatment.

To distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein-synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one-quarter and three-quarter positions in the cell was observed.

Bacterial Proteins↗
PubMed1994

Significance of plasmolysis spaces as markers for periseptal annuli and adhesion sites.

During hyperosmotic shock, the protoplast and stretched-out peptidoglycan layer first shrink together until the turgor pressure in the cell is relieved. Being non-compressible, the outer and inner membranes must fold their superfluous surfaces. While the protoplast contracts further, the inner membrane rearranges into plasmolysis spaces visible by phase-contrast microscopy. Two opposing theories predict a similar positioning of spaces in dividing cells and filaments: the 'periseptal annulus model', based on adhesion zones, involved in the predetermination of the division site; and a 'restricted, random model', based on physical properties of the protoplast. Strong osmotic shock causes retraction of the inner membrane over almost the entire surface forming the so-called 'Bayer bridges'. These tubular adhesion sites are preserved by chemical fixation, and can be destroyed by cryofixation and freeze-substitution of unfixed cells. Both the regular positioning of the plasmolysis spaces and the occurrence of tubular adhesion sites can be explained on the basis of physical properties of the membrane which necessitate rearrangements by membrane flow during shrinkage of the protoplast.

Biomechanical Phenomena↗
PubMed1994

Nucleoid partitioning and the division plane in Escherichia coli.

Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two- to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell. To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition. In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell. When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid. It thus appears that the nucleoid motive force does not depend on DNA synthesis and that cell diameter is determined not by the amount of DNA per chromosome but rather by the synthetic activity surrounding the nucleoid. Under the non-steady-state but balanced growth conditions induced by thymine limitation, nucleoids become separated into small lobules, often lying in asymmetric configurations along the cell periphery, and oblique and asymmetric division planes occur in more than half of the constricting cells. We suggest that such irregular DNA movement affects both the angle of the division plane and its position.

Cell Division↗
PubMed1993

Dimensional rearrangement of Escherichia coli B/r cells during a nutritional shift-down.

In a search for the mechanism underlying dimensional changes in bacteria, the glucose analogue methyl alpha-D-glucoside was used to effect a rapid reduction in the mass growth rate of Escherichia coli by competitively inhibiting glucose uptake, a so-called nutritional shift-down. The new steady-state cell mass and volume were reached after 1 h, during which the rate of cell division was maintained; rearrangement of the linear dimensions (cell length, diameter), however, required an additional 2 h and caused an undershoot in cell length, consistent with the view that E. coli is slow to modify its diameter. The results are compared with the overshoot in cell length that occurs following nutritional shift-up.

Cell Division↗
PubMed1993

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.

Cell Cycle↗
PubMed1993

Plasmolysis bays in Escherichia coli: are they related to development and positioning of division sites?

Plasmolysis bays, induced in Escherichia coli by hypertonic treatment, are flanked by zones of adhesion between the plasma membrane and the cell wall. To test the proposition of Cook et al. (W. R. Cook, F. Joseleau-Petit, T. J. MacAlister, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 84:7144-7148, 1987) that these zones, called periseptal annuli, play a role in determining the division site, we analyzed the positions of these zones by phase-contrast and electron microscopy. In situ treatment of cells grown in agar showed that the youngest cell pole was the most susceptible to plasmolysis, whereas the constriction site was resistant. Lateral bays occurred only at some distance from a polar bay or a resistant constriction site. Orienting cells with their most prominently plasmolyzed polar bay in one direction showed that the lateral bays were always displaced away from the polar bay at about half the distance to the other cell pole. If no poles were plasmolyzed, lateral bays occurred either in the centers of nonconstricting cells or at the 1/4 or 3/4 position of cell length in constricting cells. The asymmetric positions of lateral plasmolysis bays, caused by their abrupt displacement in the presence of polar bays or constriction sites, does not confirm the periseptal annulus model (Cook et al.), which predicts a gradual and symmetric change in the position of lateral bays with increasing cell length. Our analysis indicates that plasmolysis bays have no relation to the development and positioning of the future division site.

Cell Division↗
PubMed1992

Confocal microscopy in microgravity research.

We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

Animals↗
PubMed1992

New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways.

Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products. Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division. We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins. Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids). Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition.

Bacterial Proteins↗
PubMed1991

Identification and functional analysis of the nuclear localization signals of ribosomal protein L25 from Saccharomyces cerevisiae.

The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase. Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus. Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself. Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein. This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25. The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits. Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.

Amino Acid Sequence↗
PubMed1991

Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle.

Vacuoles of Saccharomyces cerevisiae were visualized by phase-contrast microscopy. Visualization was enhanced by adding polyvinylpyrrolidone. Vacuolar segregation during the cell cycle was analysed in 42 individual cells of strain X2180 by time-lapse photomicrography. Within 15 min of bud emergence, more than 80% of the cells contained a vacuolar segregation structure in the form of either a tubule or an alignment of vesicles. The structure emerged from one point of the mother vacuole, then elongated and moved into the bud in a few minutes. The vacuolar segregation structure disappeared, usually within 20 min, before nuclear migration, leaving a separate vacuole in the bud. To test the generality of this observation several strains were grown in the presence of the vacuolar vital dye fluorescein isothiocyanate. The bud size was used to measure progress in the cell cycle. All strains formed vacuolar segregation structures in cells with small buds, although with variations in duration and timing in the cell cycle. In the presence of nocodazole vacuolar segregation occurred normally, thus, microtubules seem not to be essential in this process.

Cell Cycle↗
PubMed1991

Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid.

Peptidoglycan synthesis rate in nonconstricting filaments of Escherichia coli dnaX(Ts) has been studied by autoradiography of incorporated [3H]diaminopimelic acid. Analysis of autoradiograms of whole cells and sacculi showed that peptidoglycan is synthesized at a reduced rate in the nucleoid-containing parts of these filaments. The lower rate of peptidoglycan synthesis in the cell center coincides with a higher local rate of protein synthesis. DNA-less cell formation in dnaX(Ts), dnaX(Ts) sfiA, and the minB minicell-forming mutant is accompanied by a local increase in peptidoglycan synthesis at the constriction site.

Autoradiography↗
PubMed1991

Amount of peptidoglycan in cell walls of gram-negative bacteria.

The amount of diaminopimelic acid (Dap) in the cell wall of Escherichia coli was measured in two ways. A radiochemical method first described by us in 1985 (F. B. Wientjes, E. Pas, P. E. M. Taschner, and C. L. Woldringh, J. Bacteriol. 164:331-337, 1985) is based on the steady-state incorporation of [3H]Dap during several generations. Knowing the cell concentration and the specific activity of the [3H]Dap, one can calculate the number of Dap molecules per sacculus. The second method measures the Dap content chemically in sacculi isolated from a known number of cells. With both methods, a value of 3.5 x 10(6) Dap molecules per sacculus was obtained. Combined with electron microscopic measurements of the surface area of the cells, the data indicate an average surface area per disaccharide unit of ca. 2.5 nm2. This finding suggests that the peptidoglycan is basically a monolayered structure.

Autoradiography↗
PubMed1990

In vivo and in vitro analysis of structure-function relationships in ribosomal protein L25 from Saccharomyces cerevisiae.

We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.

Binding Sites↗
PubMed1990

The Escherichia coli minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids.

Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.

Bacterial Proteins↗
PubMed1989

Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA.

The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants. In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random. We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.

Cell Division↗