Endocytosis and inositol hexakisphosphate levels inras transformants ofDictyostelium discoideum amoebae

Summary Fluid-phase pinocytosis kinetics and lysosomal enzyme secretion parameters were measured inDictyostelium discoideum amoebae constructed from strain AX3 by transformation with a multicopy plasmid carrying either a normalras gene (ras-Gly12), a mutatedras gene (ras-Thr12) or by the vector carrying the geneticin resistance gene only (pDNEO2). It was found that the pinocytosis rate and extent as well as the lysosomal enzyme secretion were slightly different in the three strains. These changes, however, were related to minor modifications of the cellular volumes. The overall concentration of inositol hexakisphosphate was similar in the three strains.This work was supported in part by a grant from the Ligue Nationale Française contre le Cancer (n^o 880258) to MS, and by a grant from the Fonds National Suisse de la Recherche Scientifique (No. 3.623-087) to CR.     

Retrotransposable elements in the Dictyostelium discoideum genome

Repetitive DNA is a major component of any living cell. In eukaryotes retrotransposable elements make up several percent of the genome size, and consequently, retroelements are often identified in experiments aimed at establishing physical maps and whole genome sequences. In this review, recent progress in the characterization of retrotransposable elements in the genome of the eukaryotic mi croorganism Dictyostelium discoideum is summarized with a focus on retroelements which integrate near transfer RNA genes with intriguing position specificity. Received 21 November 1997; received after revision 6 January 1998; accepted 6 January 1998     

Synergism between aggregation mutants of Dictyostelium discoideum]

The cells of an aggregateless mutant of Dictyostelium disco?deum, agip 235, can cooperate with other aggregateless or wild strains to form differentiated aggregates. A soluble mediator liberated by the coaggregating cells seems responsible for the development of agip 235. In most cases, the development of mutant agip 235 stops at the aggregation stage; however, its coaggregation with the mutant 518 results in cosporulation, with the production of viable spores of each genotype, effecting a phenotypic suppression of both mutations.     

The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum

Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum, several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium. Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.     

Regular oscillations in suspensions of a putatively chaotic mutant of Dictyostelium discoideum

We have tested the light-scattering properties of suspensions of the Dictyostelium discoideum mutant HH201 derived from the mutant Fr17. Previous studies indicated that HH201 and Fr17 possess highly irregular rhythmic properties which might represent aperiodic oscillations, i.e. chaos. We report that the former mutant can display regular oscillatory behavior. Possible explanations for this result are discussed, including that of a transition from chaotic to periodic behavior resulting from some parameter change or from strong intercellular coupling in cell suspensions.     

A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum

Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium.     

The effect of an ionophore on the aggregation of Dictyostelium discoideum]

Treatment during starvation of D. discoideum amoebae with micromolar amounts of A23187 causes an enhanced aggregation. Cells develop the properties of differentiated, aggregation competent amoebae earlier than untreated populations. Ionophore increases the release of calcium, and prevents the excretion of the phosphodiesterase ihibitor normally released in the media. A23187 suppresses the morphogenetic block of some aggregates mutants, suggesting that the ionophore either activates cAMP synthesis and excretion, or increases the cellular sensitivity to extracellular cAMP signals. This might result from the enhanced mobilisation of intracellular calcium     

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. Received 14 November 1997; received after revision 16 March 1998; accepted 16 March 1998     

Possible role of cell contact in the control of a differentiation program in Dictyostelium discoideum]

Synthesis of cAMP-phosphodiesterase falls at the end of the aggregation phase in Dictyostelium discoideum. Exogenous cAMP pulses, known to induce they synthesis of that enzyme during the course of the aggregation process, do not prevent the shut off of enzyme synthesis. Specific intercellular contacts which form at the end of aggregation seem to be required for the inhibition of enzyme synthesis.     

Change in compatibility after microinjection of cytoplasm in amoebae

Summary A cytoplasmic fraction from D32, a clone of amoebae derived fromAmoeba proteus injected with cytoplasm fromA. discoides, inhibited cell division inA. proteus but not inA. discoides indicating a permanent change with respect to compatibility.     

Molecular basis of localized responses during chemotaxis in amoebae and leukocytes

Free-living amoebae as well as mammalian leukocytes sense chemoattractants with seven helix receptors linked to G-proteins. The cells respond by extending pseudopods and moving in the direction of the highest concentration. Recent studies using GFP-tagged proteins in Dictyostelium have shown that the directional response becomes sharply localized downstream of the receptors and G-proteins but upstream of the actin cytoskeleton. These studies together with the isolation novel genes by insertional mutagenesis in Dictyostelium are leading to a new understanding of chemotaxis in eucaryotic cells.     

Regular oscillations in suspensions of a putatively chaotic mutant ofDictyostelium discoideum

Summary We have tested the light-scattering properties of suspensions of theDictyostelium discoideum mutantHH201 derived from the mutantFr17. Previous studies indicated thatHH201 andFr17 possess highly irregular rhythmic properties which might represent aperiodic oscillations, i.e. chaos. We report that the former mutant can display regular oscillatory behavior. Possible explanations for this result are discussed, including that of a transition from chaotic to periodic behavior resulting from some parameter change or from strong intercellular coupling in cell suspensions.     

Phenytoin-induced DNA synthesis and inositol 1,4,5-triphosphate formation in L-929 fibroblasts

Summary Culture of L-929 fibroblasts in the presence of phenytoin (2.5–5.0 g/ml) increased DNA synthesis, as indicated by increased [³H]thymidine uptake, while a higher dose (20 g/ml) inhibited DNA synthesis. In like manner, a low dose of phenytoin (5.0 g/ml) was effective in increasing inositol 1,4,5-trisphosphate formation while a higher dose (10 g/ml) tended to inhibit this activity. These data suggest that the formation of inositol phosphate second messengers may play a role in phenytoin-induced fibroblast proliferation and connective tissue growth.     

Phenytoin-induced DNA synthesis and inositol 1,4,5-trisphosphate formation in L-929 fibroblasts

Culture of L-929 fibroblasts in the presence of phenytoin (2.5-5.0 micrograms/ml) increased DNA synthesis, as indicated by increased [3H]thymidine uptake, while a higher dose (20 micrograms/ml) inhibited DNA synthesis. In like manner, a low dose of phenytoin (5.0 micrograms/ml) was effective in increasing inositol 1,4,5-trisphosphate formation while a higher dose (10 micrograms/ml) tended to inhibit this activity. These data suggest that the formation of inositol phosphate second messengers may play a role in phenytoin-induced fibroblast proliferation and connective tissue growth.     

Role of the inositol 1,4,5-trisphosphate receptor in early embryonic development

There is now considerable literature on the importance of phosphatidylinositol cycle activation in transducing information of various types across the plasma membrane. Though much of the data derives from studies on somatic cells, there is increasing evidence for crucial events related to development, including fertilization, cell cycle progression and dorsoventral axis formation. In this review, focus is directed mainly to the molecular basis of the inositol 1,4,5-triphosphate receptor expressed in oocytes and early embryos of Xenopus. Recent progress in studies concerning the role of this receptor in early embryonic development is discussed.