Effect of phenylarsine oxide on the fission yeast Schizosaccharomyces pombe cell cycle.

Phosphotyrosyl turnover is an essential regulatory mechanism for many biological processes, and the balance between tyrosine kinases and phosphatases plays a major role in the control of cell proliferation. Phenylarsine oxide (PAO), a potent inhibitor of tyrosine phosphatases (PTPase), was used to investigate the involvement of PTPase in the growth and control of the cell cycle of the fission yeast Schizosaccharomyces pombe. Cell proliferation was arrested by treatment with PAO, which was found to inhibit cdc25 PTPase in vitro but appeared not to act in vivo on this mitosis inducer. The PAO-treated cells displayed a mono- or binucleated phenotype and a DNA content that was either 2C or 4C, indicating a cell cycle arrest with a failure to complete cytokinesis. Entry into the cell division cycle from the G0 quiescent stage was also delayed by treatment with PAO. These results suggest that a number of key events in the mitotic cell cycle are regulated by as yet unidentified PTPases.

Characterization of thermosensitive mutants of Physarum polycephalum a plasmodial screening method for cell cycle mutants based upon synchronization at the restrictive temperature.

A screening procedure for cell cycle mutants among is mutants of Physarum has been developed for unsynchronized microplasmodia. The synchronization of the microplasmodia and the ratio of pre- to post-mitotic nuclei were evaluated after a transient shift-up to the non-permissive temperature. In contrast with wild type and is mutants not blocked in a cell cycle event, putative cell cycle mutants were synchronized. Among them, three strains which showed a reduction of DNA synthesis had an abnormally increased ratio of pre- to post-mitotic nuclei. Thus, this methodology seems to be efficient and more rapid than the previously published procedures for detecting cell-cycle mutants of Physarum.

Role of the fission yeast nim 1 protein kinase in the cell cycle response to nutritional signals.

The fission yeast cdr1/nim1 protein kinase phosphorylates and inactivates the weel cdc2-inhibitory kinase. We have investigated the role played by cdr1/nim1 in the connection between nutritional signals and the cell cycle machinery. We show that loss of nim1 activity impairs the appropriate cellular adaptation to nutritional changes. However, the reduction in cell size at division in response to nitrogen starvation is independent of nim1. Moreover, we report that nim1 is an unstable protein that is rapidly degraded upon starvation, through a mechanism that is dependent upon protein synthesis. We propose that nim1, as a constitutive indirect activator of cdc2 at mitosis, favors the cellular response to starvation but does not actively participate in it. On the contrary, upon nitrogen starvation nim1 must be actively destroyed to protect the cells from a commitment into the cell cycle under unfavourable growth conditions.

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle.

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.

[Demonstration of the action of methyl benzimidazole 2 yl carbamate (MCB) and methyl(5(2 thienyl carbonyl) 1 H benzimidazole 2 yl carbamate) (R17934) on the nucleus of Physarum polycephalum (Myxomycetes)]. / Mise en évidence de l'action du méthyl benzimidazole 2 yl carbamate (MBC) et du méthyl[5(2 thiényl carboyl) 1 H benzimidazole 2 yl carbomate] (R 17934) sur le noyau de Physarum polycephalum (Myxomycètes)

The toxicity of these compounds was determined in the amoebae and plasmodia. An electron microscopic study shows an increase in nuclear size which is in agreement with the increase of the total amount of DNA. Microtubules are present but they are not organized in a normal mitotic apparatus. Some other nuclear abnormalities are described.

Genetic analysis of the relationships between the amoebal extranuclear spindle-organizing centre and the plasmodial intranuclear spindle-organizing centre of Physarum during conjugation.

Physarum amoebae possess an extranuclear spindle-organizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.

The fission yeast Nim1/Cdr1 kinase: a link between nutritional state and cell cycle control.

Close connections appear to exist between extra-cellular signals that regulate cell proliferation and the protein kinases that control the cell cycle machinery. The fission yeast nim1 kinase is an inducer of cdc2 kinase activity acting through the inhibition of wee1 kinase. Nim1 function is required for a correct cellular response to nutritional starvation. In the absence of nim1, starved cells are unable to decrease their size at mitosis, to arrest their cycle in G1 and to enter G0. Here, we review our current knowledge on the role and the regulation of nim1 in connecting cell cycle and nutritional pathways.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum.

The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. The mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cytoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.