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1.
Bestatin methyl ester (BME) is an inhibitor of Zn(2+)-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn(2+)-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA.  相似文献   

2.
A genetic melanotic neoplasm of Drosophila melanogaster   总被引:6,自引:0,他引:6  
The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.  相似文献   

3.
We describe rblA, the Dictyostelium ortholog of the retinoblastoma susceptibility gene Rb. In the growth phase, rblA expression is correlated with several factors that lead to 'preference' for the spore pathway. During multicellular development, expression increases 200-fold in differentiating spores. rblA-null strains differentiate stalk cells and spores normally, but in chimeras with wild type, the mutant shows a strong preference for the stalk pathway. rblA-null cells are hypersensitive to the stalk morphogen DIF, suggesting that rblA normally suppresses the DIF response in cells destined for the spore pathway. rblA overexpression during growth leads to G1 arrest, but as growing Dictyostelium are overwhelmingly in G2 phase, rblA does not seem to be important in the normal cell cycle. rblA-null cells show reduced cell size and a premature growth-development transition; the latter appears anomalous but may reflect selection pressures acting on social ameba.  相似文献   

4.
Upon starvation, Dictyostelium amoebae aggregate together and then differentiate into either the stalk or spore cells that, respectively, form the stalk and sorus of the fruiting body. During differentiation, the prestalk and prespore cells become spatially segregated in a clearly defined developmental pattern. Several low molecular weight molecules that influence cell type determination during in vitro differentiation have been identified. The possible role of these molecules as morphogens, responsible for the formation of the developmental pattern, is discussed.  相似文献   

5.
The DIFs are a family of secreted chlorinated molecules that control cell fate during development of Dictyostelium cells in culture and probably during normal development too. They induce stalk cell differentiation and suppress spore cell formation. The biosynthetic and inactivation pathways of DIF-1 (the major bioactivity) have been worked out. DIF-1 is probably synthesised in prespore cells and inactivated in prestalk cells, by dechlorination. Thus, each cell type tends to alter DIF-1 level so as to favour differentiation of the other cell type. This relationship leads to a model for cell-type proportioning during normal development.  相似文献   

6.
Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.  相似文献   

7.
Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.  相似文献   

8.
We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.  相似文献   

9.
The choice of the stalk cell differentiation pathway in Dictyostelium is promoted by an endogenous substance, DIF-1, which is 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone. It is also favoured by weak acids and two inhibitors of the plasma membrane proton pumps of fungi and plants, diethylstilbestrol (DES) and zearalenone, and antagonised by ammonia and other weak bases, which promote spore differentiation. These observations led to the proposal that the choice of differentiation pathway is regulated by intracellular pH. They also prompted the conjecture that DIF-1 itself is a plasma membrane proton pump inhibitor. We report here experiments showing that DIF-1 is not a plasma membrane proton pump inhibitor. We demonstrate that diethylstilbestrol and zearalenone do inhibit the plasma membrane proton pump of Dictyostelium and we show that there is an excellent qualitative and quantitative correlation between the inhibitory activity of these agents, and of a number of other substances, and their ability to divert differentiation from the spore to the stalk pathway. We conclude that inhibition of the plasma membrane proton pump does shift the choice of differentiation pathway in Dictyostelium towards the stalk pathway, but that DIF does not act by this route, and we propose a model for the actions of DIF and plasma membrane proton pump inhibitors in which the differentiation pathway is controlled by the pH of intracellular vesicles rather than by intracellular pH itself. The model invokes a DIF- and proton-activated vesicular chloride channel whose opening permits acidification of the vesicles and lowers cytosolic Ca++ concentration.  相似文献   

10.
Free-living amoebae of the cellular slime mouldDictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case ofDictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) ‘coin tossing’, whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) ‘chemical kinetics’, whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) ‘positional information’, in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arisesin situ  相似文献   

11.
12.
A mutant of Dictyostelium discoideum, HM18, will differentiate into both stalk and spore cells when plated at high cell density (105 cells/cm2) as a monolayer on non-nutrient agar containing 5 mM cAMP [6]. At low cell density (103 cells/cm2) neither stalk nor spore cells are produced, but the addition of a cytosol fraction leads to stalk cell formation, and the addition of a membrane fraction leads to spore cell formation. The spore cell-inducing activity of the cell membranes is developmentally regulated; it is first detectable during late aggregation and increases to a maximum level in the pseudoplasmodial stage of development. The activity is sensitive to proteolysis and insensitive to periodate treatment. It is partially inactivated by incubation at 100 °C for 5 min. Variable amounts of the activity can be removed from the membrane by washing, suggesting that at least part of the activity is loosely membrane-bound. Activity is enriched in plasma membrane fractions, suggesting that the inducing factor is located at the cell surface. It is possible that the membranes are replacing a cell-cell contact requirement for spore formation.  相似文献   

13.
14.
Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.  相似文献   

15.
The P4 variant of Dictyostelium discoideum is characterized by the production of fruiting structures in which the overall proportion of stalk to spore material is increased, relative to the wild type. The altered morphology of the mutant is due to increased sensitivity to cyclic AMP which promotes stalk cell differentiation. In the presence of 10-4 M-cyclic AMP the entire population of P4 amoebae forms clumps of stalk cells on the surface of the dialysis membrane support. Measurement of changes in activity of a range of developmentally-regulated enzymes during the development of P4 in the presence and absence of cyclic AMP has allowed us to identify three classes of enzyme: (i) Those, such as beta-glucosidase II, trehalose-6-phosphate synthetase and uridine diphosphogalactose-4-epimerase, which are required for the production of spores. (ii) Enzymes, primarily but perhaps not exclusively, required during stalk cell formation. Typical of these are N-acetylglucosaminidase and alkaline phosphatase. (iii) General enzymes, such as threonine dehydrase, alpha-mannosidase and uridine diphosphoglucose pyrophyosphorylase, which are present inboth pre-stalk and pre-spore cells and appear to be necessary for the development of both cell types.  相似文献   

16.
We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.  相似文献   

17.
We describe a method of separating prestalk and prespore cells of Dictyostelium discoideum slugs using a self-generating Percoll gradient. This method gives quantitative recovery of cells and good purity. Separated prestalk and prespore cells possess different levels of the enzymes UDP galactose :polysaccharide transferase, cAMP phosphodiesterase and glycogen phosphorylase. We have used this method, as well as mechanical dissection of slugs, to examine the fate of separated prestalk and prespore cells in Dictyostelium strains that are able to give rise to mature stalk and spore cells in cell monolayers. The results from such experiments provide direct evidence that prestalk and prespore cells from the migrating slug stage are programmed to differentiate into stalk and spore cells respectively.  相似文献   

18.
The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.  相似文献   

19.
Single-celled myxamoebae undergo differentiation into either stalk cells or spore cells during a 24-hr period in Dictyostelium discoideum. This study employed ultramicrochemical techniques and enzymatic cycling to assess the presence of cell-specific events in spore and stalk cells. Freeze-dried sections of one organism were assayed in 0.1 μl of reaction mixture. This method was used to determine the extent of localization of trehalose in spore cells and stalk cells during development.Trehalose was low in the early stages of differentiation to about 20 hr when the level started to increase. In developing spore cells, the trehalose level increased sixfold during the last 5 hr of development. Likewise, the entire stalk contained trehalose when the stalk was first formed. At mature sorocarp, trehalose levels were the same in spores and the apex of the stalk. There was a decreasing gradient of trehalose down the stalk. The bottom one-fourth of the stalk was devoid of this disaccharide. Therefore, trehalose was degraded from an area of the stalk where it was localized earlier in development.The results of this investigation negate the assumption that trehalose is never present in the stalk. Although trehalose was found in spore cells, prestalk cells also contained high trehalose levels. The stalk cell-specific trehalose was not retained during differentiation, however, but was apparently degraded in the mature stalk cell.  相似文献   

20.
Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.  相似文献   

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