A stalk-specific localization of trehalase activity in Dictyostelium discoideum.

By utilizing ultra-microtechniques, trehalase activity was followed in specific cell types during the differentiation cycle of Dictyostelium discoideum. When whole organisms were assayed, trehalase activity was found to be high in the early stages of differentiation, decreased to its lowest point at 14 h, and then increased at the end of the cycle. By microdissection of freeze-dried individuals, the activity of trehalase could be followed during the migration of pre-stalk and pre-spore cells. No activity was observed at any stage of spore cell development, whereas stalk cells showed a rapid increase in activity upon maturation. An increasing gradient of activity was found from the apex of the stalk toward the base. This localization of trehalase in stalk cells resolves some contradictory results in the literature concerning the role of the enzyme during differentiation.     

Localization of glycogen synthetase during differentiation of presumptive cell types in Dictyostelium discoideum.

Ultramicrochemical techniques were utilized to assay glycogen synthetase (EC 2.4.1.11) activity in cell samples of Dictyostelium discoideum as small as 0.01 mug (dry weight) in reaction volumes of 0.1 mul. The activity was assayed by an amplification procedure employing the enzymatic cycling of pyridine nucleotides. These techniques were used to determine the extent of localization of glycogen synthetase in the two cell types during differentiation of D. discoideum. Localization studies in developing spore cells revealed decreasing enzyme activity to the culmination stage. During this phase of development, the enzyme required the presence of soluble glycogen for activity. From culmination to sorocarp stage, enzyme activity increased and was independent of the soluble glycogen. In developing stalk cells, synthetase showed a decreasing gradient of activity. In sorocarps, the cells in the stalk apex showed synthetase activity similar to that of the spores. The cells at the bottom of the stalk had no detectable activity.     

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum

Abstract. We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the pre-stalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.     

Localization of glycogen phosphorylase in specific cell types during differentiation of Dictyostelium discoideum

The localization of glycogen phosphorylase was studied during the differentiation of prespore and prestalk cells in Dictyostelium discoideum. Ultramicrotechniques were utilized to assay the enzyme activity in cell samples as small as 0.02 μg dry wt in reaction volumes of 0.1 μl. The activity was assayed using an amplification procedure employing the enzymatic cycling of pyridine nucleotides. Glycogen phosphorylase from individual organisms was assayed during the developmental period. Early in development, activity was low but gradually increased to a maximum value at culmination. From culmination to sorocarp, enzyme activity decreased rapidly. Cell-specific assays of spores showed that phosphorylase activity increased slightly to culmination, and then decreased. Prestalk cells showed the greatest activity in the area of stalk sheath construction and elongation. Stalk cells showed a decreasing gradient of enzyme activity from the tip of the stalk to the base. Enzyme activity in the spores may be sufficient to provide glucose units for trehalose synthesis and spore coat production. The prestalk enzyme may degrade glycogen to provide glycosyl units for production of the stalk sheath and trehalose. Possible models of cell-specific biochemical events in Dictyostelium discoideum are discussed.     

Possible cooperation of differential adhesion and chemotaxis in mound formation of Dictyostelium.

In the mound stage of Dictyostelium discoideum, pre-stalk cells sort and form a tip at the apex. How this pattern forms is as yet unknown. A cellular level model allows us to simulate both differential cell adhesion and chemotaxis, to show that with differential adhesion only, pre-stalk cells move to the surface of the mound but form no tip. With chemotaxis driven by an outgoing circular wave only, a tip forms but contains both pre-stalk and pre-spore cells. Only for a narrow range of relative strengths between differential adhesion and chemotaxis can both mechanisms work in concert to form a tip containing only pre-stalk cells. The simulations provide a method to determine the processes necessary for patterning and suggest a series of further experiments.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

Abstract. The effects of migration and culmination on patterning of presumptive (prespore and prestalk) cells and mature (spore and stalk) cells of D. discoideum were investigated. The ratio of prespore to total cells, as determined by staining with fluorescein-conjugated antispore globulin, was constant (77%) up until 8 h of slug migration, but then decreased to a level (64%) which thereafter remained unchanged during migration. Cells which lost prespore antigen during migration were located in the posterior (prespore) part next to the agar surface.
Upon induction of culmination, however, the ratio of prespore cells quickly increased to the normal level (77%) within 1–2 h. During the transition between migration and culmination prestalk and prespore cells were considerably intermixed within the cell mass, before the normal prestalk-prespore pattern was reestablished at the preculmination (Mexican hat) stage. Spore: stalk ratios within fruiting bodies were normal irrespective of the lengths of slug migration.     

Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium

Abstract. Depending upon environmental conditions, developing cells of the cellular slime mold Dictyostelium discoideum may enter a slug stage in which the cell mass migrates in response to gradients of light and temperature. This developmental stage has often been used to study the divergent differentiation of the cells that will subsequently form spores and stalk in the mature fruiting body. However, still debated is the extent to which the differentiation evident in slug cells is a precondition for development of the mature cells in fruits. Using two-dimensional gel electrophoresis of polypeptides, we have examined the proteins made by prespore and prestalk cells of migrating slugs and by maturing spore and stalk cells. The data indicate that many of the cell-type specific polypeptides in prespore cells of slugs persist as cell-type specific polypeptides of mature spores. Prestalk slug cells, in contrast, do not contain significant amounts of stalk-specific proteins; these proteins appear only during culmination. The precursor cell types also differ in the times and rates of synthesis of cell-specific proteins: prestalk proteins appear much earlier in development than do the prespore, but never reach the levels of expression that the prespore proteins do later in culmination. These findings may explain the well established ability of prespore cells to regulate their cell type more rapidly than do prestalk cells. There are also implications for our general understanding of what is a 'prestalk' gene product.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.     

ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum

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.     

Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum

Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.     

Cell specific activity of trehalose-6-phosphate synthetase during differentiation of Dictyostelium discoideum.

Trehalose-6-P synthetase activity was low at the beginning of the life cycle of Dictyostelium discoideum, reached maximum activity at 20 h, and decreased at late sorocarp. Enzyme activity in developing spore cells increased 10-fold during differentiation from myxamoebae (0 h) to the culmination stage (20 h) and decreased slightly at sorocarp (24 h). Activity was similar in spore cells at the apex of the stalk. The activities in the stalk cells were dependent upon their position in the developing stalk. There was a decreasing gradient of activity from the apex to the base of the stalk.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。     

The organisation of fruiting body formation in Dictyostelium minutum

The process of culmination was investigated in three strains of the species Dictyostelium minutum. After aggregates have been formed a pulsatile signalling mechanism arises; the centre of signal emission becomes the apex of the developing fruiting structure. In the late aggregate, all cells differentiate into prespore cells. Cells that have reached the apex of the culminating cells mass redifferentiate into stalk cells. In two of the three D. minutum strains, interruption of regular stalk formation, more or less random formation of stalk cells and the synthesis of stalk supporting material from cell debris often takes place. The formation of multiple apices on aggregates and early fruiting structures is characteristic for these two strains. Within the species D. minutum, the exhibition of a marked pulsatile signalling mechanism is correlated with a capacity to form a regularly shaped stalk and to organize relatively large cell masses. The possible function of pulsatile signalling in the culmination process is discussed.     

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

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.     

Quantitative analysis of the spatial distribution of ultrastructural differentiation markers during development ofDictyostelium discoideum

Summary The appearance and spatial distrubution of ultrastructural markers ofDictyostelium discoideum differentiation were quantitatively analysed. Our results combined with data from the literature on the functions of cells at various stages of development lead to the following conclusions. When food is no longer available all amoebae initially develop an autophagic apparatus in order to sustain metabolism. After slugs have been formed, autophagy is suppressed in the prespore cells. During aggregation a number of cells gradually form prespore characteristics. These cells arise at random but later they become located in the basal part of the tip-forming aggregate. From the early slug stage onwards, cells of the posterior two third region gradually enter into the prespore pathway. During prolonged slug migration the optimal acquirement of prespore characteristics is blocked. Cells of the anterior region show no active differentiation but they maintain the morphology and most of the functions of aggregating cells. At the rear-guard of the slug and later on in the basal region of the maturing fruiting body, a second anteriorlike region appears. Actual stalk cell differentiation takes place only at the apex and at the base of the developing fruiting body.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

The timing of cell-type-specific differentiation in Dictyostelium discoideum

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.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.