Graeme M. Walker

Cell cycle specificity of certain antimicrotubular drugs in schizosaccharomyces pombe

Of the seven antimicrotubular drugs tested, nocodazole, mebendazole and trifluralin at saturable concentrations failed to inhibit cell division in Schizosaccharomyces pombe, while carbendazim, thiabendazole and chloropropham each at 50 micrograms ml- and amiprophos methyl at 200 micrograms ml-1 completely arrested cell division. This inhibition was associated with striking morphological changes in which carbendazim- and thiabendazole-treated cells became elongated and pseudohyphal, whereas chloropropham- and amiprophos methyl-treated cells appeared small and rounded with occasional V-shaped pairs. Lomofungin staining revealed that nuclear division was also arrested by these drugs. Suspected blockage of defined cell cycle stages was confirmed by pulse-induction experiments which revealed that cells could be synchronized into division using exposure to a drug for one generation. Further experiments with synchronous cultures prepared by size selection showed that different drugs possessed different transition points; for example, carbendazim and thiabendazole were effective in blocking a late stage of the cell cycle just prior to division, whereas amiprophos methyl affected a very early stage. The results suggest that some of the drugs used exert cell cycle specificity in S. pombe either by impairing microtubule assembly mechanisms (as with carbendazim and thiabendazole) or by inhibiting synthesis of tubulin subunits (as with amiprophos methyl). These drugs could prove useful in studies of microtubule biogenesis during the cell cycle in yeast.

Magnesium and the regulation of germ-tube formation in candida albicans

Candida albicans requires Mg2+ for germ-tube formation. Mg-deficient media, metal ion chelators and the ionophore A23187 inhibited germ-tube formation. Cell Mg content during exponential yeast-phase growth remained constant but increased throughout germ-tube formation. The onset of germ-tube formation coincided with a sharp peak in Mg concentration within the cells. Yeast-phase cells of strain CA2, which did not form germ-tubes, had a lower Mg content and failed to accumulate Mg when incubated under conditions for germ-tube formation. Mg also increased the uptake and incorporation of N-acetylglucosamine. These findings point to a central regulatory role for Mg in C. albicans morphogenesis.

Magnesium-induced mitochondrial polymorphism and changes in respiratory metabolism in the fission yeast, Schizosaccharomyces pombe

The role of magnesium ions in controlling mitochondrial structure and function in the fission yeast, Schizosaccharomyces pombe, has been studied. Results show that in control cultures of S. pombe grown in the presence of magnesium, cells follow predominantly a fermentative pathway of metabolism. The mitochondria, as visualized by electron microscopy, are large but few in number, and appear irregularly branched with indistinct cristae. On depletion of the magnesium supply, cell division ceases after about 4 hours after which there is an increase in respiration. The mitochondria increase in number and become small with well-defined cristae. On replenishment of magnesium ions mitochondria seem to fuse and there is a gradual restoration of the respiratory metabolism characteristic of control cells.

An Investigation into the Potential Use of Chelating Agents and Antibiotics as Synchrony Inducers in the Fission Yeast Schizosaccharomyces pombe

Summary: Following the discoveries that the divalent cation ionophore A23187 and the divalent cation chelating agent EDTA can be used to synchronize yeast cell division, a study has been undertaken of the possible use of other chelating agents and antibiotics which interact with divalent cations in controlling cell division in the fission yeast Schizosaccharomyces pombe. All the agents studied (five chelators and two antibiotics) arrested cell division in growing cultures of this yeast, but only sodium pyrophosphate and citrate induced synchrony of cell division. Novobiocin produced a transient inhibition of cell division, treated cells exhibiting “endogenous recovery” in the continued presence of the antibiotic. The results obtained are discussed in relation to the hypothesis that the concentration of intracellular Mg2+ regulates cell division.

Synchronization of Cell Division in the Fission Yeast Schizosaccharomyces pombe by Ethylenediaminetetra-acetic Acid

Summary: When cultures of Schizosaccharomyces pombe growing exponentially in a minimal medium were treated with 50 mM-EDTA for 1 h, nucleic acid synthesis was inhibited but protein synthesis was not. On transfer to fresh medium, 50% of the cells divided synchronously. These phenomena may be explained on the basis of reduced availability of Mg2+ following chelation with EDTA.

Changes in calcium and magnesium levels during heat-shock synchronized cell division in Tetrahymena.

Abstract Calcium and magnesium contents were measured in cells of Tetrahymena pyriformis induced to divide synchronously by a multi-heat-shock procedure. During free-running synchronized cell division in complex proteose peptone medium, significant peaks of both calcium and magnesium were observed at points in the cell cycle just prior to division. No such peaks were detected in cells dividing asynchronously in proteose peptone. When synchronized cell division was followed after transfer to an inorganic medium, cell calcium and magnesium levels were observed to decrease in relation to the corresponding cell number increase, indicating that in concentration terms, calcium and magnesium remain fairly constant. This latter result suggests that neither calcium nor magnesium influxes act as triggers for cell division in Tetrahymena and that the fluctuations of these metals seen during the synchronized division cycle in complex medium represent an effect rather than a cause.

THE REGULATION OF CELL DIVISION IN YEAST BY MAGNESIUM

ABSTRACT Cells of the fission yeast, Schizosaccharomyces pombe, and the budding yeast, Kluyveromyces fragilis, fail to divide in Mg-free minimal media but can be induced to divide synchronously on restoration of Mg to “exhausted” cultures. Mg-depleted S. pombe, cells appear abnormally long, lack cell plates and possess elongated nuclei. This, together with evidence of a late cell cycle arrest by the ionophore A23187, suggests that specific Mg limitation blocks cells late in the G2 phase of the cell cycle. Moreover, analysis of cell Mg in cultures synchronized by various techniques reveals that there is a fairly steady fall in Mg concentration as cells grow, terminating in a rapid influx of Mg just before division. These results lead to the hypothesis that intracellular Mg concentration is the transducer for size and consequently time, related control of the cell cycle. KEYWORDS Yeast cell cycle;, magnesium and cell division control;, Schizosaccharomyces pombe/Kluyveromyces fragilis synchronous division.

Correlation of glutamine synthetase activity with cell magnesium concentration during cell division in yeast synchronized by induction

Summary: Synchronization of cell division in the fission yeast Schizosaccharomyces pombe and the budding yeast Kluyveromyces fragilis was achieved by induction using the DNA synthesis inhibitor 2′-deoxyadenosine and by a magnesium-exhaustion technique. The activity of glutamine synthetase in these synchronized cultures oscillated. Variations in the intracellular magnesium concentration were also observed, and peaks in magnesium concentration correlated with peaks in enzyme activity. We suggest that the enzyme from yeast is unstable and that its activity is regulated in vivo by changes in the intracellular concentration of magnesium.