Ultrastructural Changes of the Two Types of Differentiated Cells during the Migration and Early Culmination Stages of Dictyostelium discoideum

Changes of fine structure during prolonged migration of Dictyostelium discoideum slugs were studied by electronmicroscopy. Prespore specific vacuoles of cells located near the substratum gradually degenerated and the prespore antigen contained in them was lost. During the process, mitochondria in the prespore cells were transformed dramatically: as the mitochondrion elongates, its central part becomes thinner and the cristae become localized at its two ends. Then it bends and its two ends fuse to segregate part of the cytoplasm. The cristae then accumulate in the original ends. Similar mitochondrial transformation was observed in prespore cells of cell masses induced to culminate after a long period of migration.     

Cell Differentiation in Dictyostelium discoideum

We have shown previously that amoebae of D. discoideum strain V12 M2 starved at low density in the presence of cyclic AMP fail to form either stalk cells or prespore cells; a low molecular weight factor released by cells at high density promotes stalk formation under these conditions but formation of prespore cells requires 'cell contact'. Here we summarise evidence that:
1. Elevated intracellular cyclic AMP levels are required for all developmental gene expression beyond the preaggregative phase, and ammonia antagonises this expression in some way. However, the action of ammonia is not pathway specific.
2.'Cell contact' is a specific requirement for entry into the prespore pathway of gene expression since isolated cells provided with cyclic AMP synthesise much reduced amounts of the presporespecific enzyme uridine diphosphate (UDP) galactose polysaccharide transferase but normal amounts of the pathway-indifferent enzyme glycogen phosphorylase.
3. The 'cell contact' mechanism is uniquely sensitive to low concentrations of pronase. This protease selectively inhibits transferase synthesis and blocks in vitro spore differentiation (in a spore-forming mutant). It does not prevent chemotactic aggregation, stream formation, or stalk cell formation in the presence of cyclic AMP.     

Cell Migration: Regulation of cytoskeleton by Rap1 in Dictyostelium discoideum

Cell movement involves a coordinated regulation of the cytoskeleton, F-actin-mediated protrusions at the front and myosin-mediated contraction of the posterior of a cell. The small GTPase Rap1 functions as a key regulator in the spatial and temporal control of cytoskeleton reorganization for cell migration. This review outlines the establishment of cell polarity by differential localizations of the cytoskeleton and discusses the spatial and temporal regulation of cytoskeleton reorganization via the Rap1 signaling pathway during chemotaxis with a focus on recent advances in the study of chemotaxis using a simple eukaryotic model organism, Dictyostelium discoideum.     

Cell Volume Determinations of Dictyostelium discoideum

The intercellular water present in pellets of centrifuged cell suspensions of the slime mold Dictyostelium discoideum was measured at four stages of differentiation.     

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 .     

Cell Movements and Mechanical Force Distribution During the Migration of Dictyostelium Slugs

Migration of Dictyostelium discoideum slugs results from coordinated movement of their constituent cells. It is generally assumed that each cell contributes to the total motive force of the slug. However, the basic mechanisms by which mechanical forces (traction and resistive forces) are transmitted to the substrate, their magnitude and their location, are largely unknown. In this work, we performed detailed observations of cell movements by fluorescence microscopy using two-dimensional (2D) slugs. We show that 2D slugs share most of the properties of 3D ones. In particular, waves of movement propagate in long 2D slugs, and slug speed correlates with slug length as found in 3D slugs. We also present the first measurements of the distribution of forces exerted by 2D and 3D slugs using the elastic substrate method. Traction forces are mainly exerted in the central region of the slug. The large perpendicular forces around slug boundary and the existence of parallel resistive forces in the tip and/or the tail suggest an important role of the sheath in the transmission of forces to the substrate.     

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.     

Ultrastructural Changes During Germination of Dictyostelium discoideum Spores

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Role of Cell Sorting in Pattern Formation in Dictyostelium discoideum

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum , labeled cells grown in the absence of glucose [G(–) cells] and unlabeled cells grown in its presence [G(+) cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(–) cells, during formation of a standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(–) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.     

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans

The prestalk and prespore cells from the Dictyostelium discoideummulticellular slug stage of development differ in assembly ofglycoconjugates. Prespore cells are 2- to 3-fold more activethan prestalk cells in the assembly of N-linked glycans and20-fold more active in their fucosylation. Only prespore cellssynthesize an O-linked glycan consisting in part of Fuc -linkedto N-acetylglucosamine. Incorporation of fucose, glucosamine,mannose and galactose into large pronase-resistant glycoconjugateswas almost exclusively into prespore cells. Such glucosamine-labelledglycoconjugates resist fragmentation by ß-eliminationand include a glycoantigen dependent on the modB genetic locus.In contrast, large fucose-labelled glycoconjugates consistedof multiple, small, O-linked oligosaccharides on carrier peptides.The spore coat protein SP96 has several fucosylated O-linkedoligosaccharides, one of which correlates with a fucose epitopepreviously shown to localize in prespore vesicles and the outerlayer of the spore coat. Dictyostelium discoideum glycoconjugates glycoproteins prespore prestalk     

Cell growth and morphology of Dictyostelium discoideum in space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiation-sensitive mutant and the parental wild-type strain, were used to investigate the effects of microgravity and/or cosmic radiation on their morphology through the whole life span from spores to fruiting bodies for about 7 days in space shuttle of NASA. We found almost no effect of space environment on amoeba cell growth in both strains. It was also observed that almost the same number and shape of fruiting bodies in space compared to the control experiments on earth. These results suggest that there is little effect of microgravity and space radiation on germination, cell aggregation, cell differentiation and cell morphology in the cellular slime mold.     

Cell sorting within the prestalk zone of Dictyostelium discoideum

Abstract. The prestalk zone of slugs of Dictyostelium discoideum has been shown to contain three subregions in which the extracellular matrix genes ecmA and ecmB are differentially expressed; it is generally thought that these regions are defined by extracellular signals. Using β-galactosidase as a cell marker, we have shown that cells can sort specifically to all three regions. Cells from the posterior-prestalk zone ("prestalk 0 zone") which are injected into the slug tip move within 60 min back to their position of origin. When cells from the anterior prestalk zone (presumably containing a mixture of ecmA and ecmB expressers) are transplanted to the posterior prestalk zone, they move to the tip ("prestalk A zone") within 1 h and about 30 min subsequently are often found in a cone-shaped region within the tip ("prestalk B zone"). Cells transplanted to their own positions do not move significantly within this period. Since the sub-regions of the prestalk zone can be defined by sorting, it is possible that they are normally formed in this way rather than by position-dependent signals. Cells transplanted without a change in anterior-posterior position and cells which have sorted back to their positions of origin eventually spread out throughout the prestalk zone. This suggests that sorting preferences of cells are respecified. When posterior prestalk cells are transplanted to the prespore zone, respecification of sorting preference is suspended until the cells return to the prestalk zone and anterior-prestalk cells acquire posterior-prestalk sorting preferences.     

盘基网柄菌细胞发育过程中尿囊酸酶的亚细胞定位

利用胶体金免疫电镜技术,观察了盘基网柄菌细胞分化与凋亡过程中胞内尿囊酸酶的位置变化。结果表明,在细胞聚集期细胞产生的尿囊酸酶主要分布于线粒体及周围细胞质内。到了细胞丘时期,尿囊酸酶只特异地存在于发生内自噬的线粒体内,且仅局限于线粒体因内自噬产生的空泡区域,这些发生线粒体内自噬的细胞将分化成前孢子细胞。随着前孢子细胞分化的进行,尿囊酸酶颗粒在细胞内分布逐渐减少,在靠近质膜处的空泡内还能观察到一些酶颗粒;而另一些细胞内,几乎所有的胞器内都能观察到酶颗粒,一直延续至柄细胞形成。从中可以看到尿囊酸酶在将发育成孢子细胞和柄细胞两种类型细胞内的分布位置明显不同,结果提示了尿囊酸酶蛋白与盘基网柄菌细胞分化和凋亡调控途径有密切关系。     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum ), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum .     

盘基网柄菌(Dictyostelium discoideum)突变型细胞allC细胞周期异常的研究

细胞计数和细胞倍增时间计算的结果表明allC细胞的倍增时间为2.36h,仅为KAx-3细胞倍增时间的1/3。为了探究allC细胞倍增时间大幅度缩短、细胞周期异常的原因我们采用流式细胞术测定两种细胞的细胞周期,并结合实时荧光定量PCR技术测定cycB1和cdk1基因的相对表达量的比值。结果表明,16h突变型allC细胞处于G2期的数目(1.51%)显著少于KAx-3细胞(16.61%)。allC细胞和KAx-3细胞的细胞周期素B1(cyclinB1)cycB1基因相对表达量分别是2.5和0.25,两者相差10倍。这些数据表明,两种类型细胞中G2期的差异十分明显,cyclinB1的相对表达量也存在显著差异。提示cyclinB1的过表达可能在一定程度上影响allC细胞的细胞周期正常的调控机制,与突变细胞的G2期异常有一定关系。