A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Rac regulation of chemotaxis and morphogenesis in Dictyostelium

Chemotaxis requires localized F-actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F-actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant-mediated F-actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k1/2 null cells and wild-type cells treated with LY294002 exhibit a significantly reduced second peak of RacB activation, which is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F-actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F-actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F-actin polymerization, and chemotaxis in Dictyostelium.     

The determination of spatial pattern inDictyostelium discoideum

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     

阿拉伯海假交替单胞菌N1230-9两个甲基受体趋化蛋白的功能鉴定

【目的】作为海洋中的特有及优势种群,假交替单胞菌(Pseudoalteromonas)普遍拥有多个甲基受体趋化蛋白(methyl-accepting chemotaxis protein, MCP),探究这些趋化受体的功能。【方法】以太平洋表层海水来源的一株阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9为研究对象,利用软琼脂平板法测试该菌株对23种碳源的趋化能力,继而利用同源重组策略构建2个含sCache结构域MCP编码基因(woc28264和woc27036)缺失突变体,并分析突变体对10种碳源的趋化能力。【结果】菌株N1230-9对海藻糖、麦芽糖、蔗糖、N-乙酰氨基葡萄糖、l-苹果酸、乙酸钠、丙酸钠、丙酮酸钠、柠檬酸和琥珀酸10种碳源具有趋化能力。WOC28264是l-苹果酸和蔗糖的特异性趋化受体,WOC27036则是柠檬酸和琥珀酸的特异性趋化受体。此外,WOC28264和WOC27036还均是N-乙酰氨基葡萄糖和海藻糖的趋化受体。【结论】WOC28264和WOC27036存在重叠的碳源效应物。     

Unique Colony Housing the Coexisting Escherichia coli and Dictyostelium discoideum

Two well-characterized and phylogenetically diverse species, Escherichiacoli and Dictyostelium discoideum, were used as the modelorganisms. When the two species were mixed and allowed to grow onminimal agar plates at 22 ^°C, instead of the predator Dictyostelium exterminating E.coli, the two species remarkablyachieved a state of stable coexistence in about two weeks. In addition, theemerged colonies housing the coexisting species have a mucoidal naturethat is distinctive from its origin. The simplicity of the system and the shorttime span for the two species to develop the coexistence state, that isproven stable and reproducible on laboratory conditions, hence, providesa new model system for the study of symbiosis, particularly with referenceto the initial stages.     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentration of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP phosphodiesterase revealed that incomplete hydrolysis of cyclic AMP was not the reason for the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans北大核心CSCD

［Objective］ Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata.Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses.Chemotaxis appears crucial for the growth of A.caulinodansin complicated environment and the construction of associative relationship with the plant.However, little is known about the chemotactic pathway of A.caulinodans.Thus, our study aimed to compare the chemotaxis-like genes of A.caulinodans with those of well-studied species.［Methods］ NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A.caulinodans.HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein （MCP）.［Results］ There was a major chemotaxis cluster in A.caulinodans and the CheR methylated MCPs independently of pentapeptide motif.There were 43 MCP homologs containing diverse signal-sensing architectures in A.caulinodans.In addition,cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.［Conclusion］ Despite the extremely high homology presented between the chemotactic system of A.caulinodans and those of well-studied species, A.caulinodans shows its own unique characteristics.The classification of these chemotactic pathways by comparative genomics enables us to better understand how A.caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.     

Inhibition of aggregation by light in the cellular slime mold Dictyostelium discoideum

Aggregation of Dictyostelium amoebae is inhibited by light. White light intensities 10² W · cm^-2 cause an inhibition which reaches a saturation at 2 · 10³ W · cm^-2. The action spectrum, based on photon fluence-response curves, shows a major peak around 405 nm and extends through most of the visible spectrum with a secondary maximum at about 530 nm. The action spectrum of the inhibition of aggregation resembles the action spectrum of accumulations of amoebae in light traps and the action spectrum of photodispersal from light traps; it does not resemble the action spectrum of phototaxis in pseudoplasmodia.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

The effect of altered glycosylation on the characteristics of lysosomal trehalase in Dictyostelium discoideum

The trehalase I of Dictyostelium discoideum exhibits characteristics of a typical lysosomal enzyme. The enzyme is glycosylated and carries a number of negatively charged components which cause it to be a very acidic protein. Strain M31, bears a recessive mutation mod A which alters the post-translational modification of several lysosomal enzymes including trehalase. A direct consequence of this mutation is a reduction of the negatively charged components on lysosomal enzymes. This reduction in negativity is observed in the altered chromatographic and electrophoretic behaviour of M31 trehalase.Trehalase I is synthesized during spore germination. Tunicamycin prevents the formation of recoverable trehalase from germinating spores but does not interfere with the germination process. These results indicate that the trehalase I synthesized during spore germination is not required for the successful completion of spore germination. Minor modification in the glycosylation, as seen in strain M31, does not affect the enzymatic activity. However, when glycosylation is greatly reduced by tunicamycin the enzyme is inactive.     

Ammonia uptake and retention in some cyanobacteria

The internal pool of ammonia in strains of unicellular and filamentous cyanobacteria was found to be 6–12 nmol·mg^-1 protein. In nitrate grown Anacystis nidulans R-2 the pool size averaged 12 nmol·mg^-1 protein, which corresponds to 2.3 mM, and was little affected by N-source or medium pH during growth. Cells from NH ₄ ⁺ -limited continuous culture contained comparable pools, and cell yield was independent of medium pH (7.2–8.5). The internal pool was not bound to macromolecules. The pool fell transiently to about one-third within 2 h on shifting cells to N-free medium, but was slowly regenerated over 24 h.Added ammonia was removed from solution by illuminated cell suspensions at a linear rate, adequate to supply biosynthetic needs, to residual concentrations less than 5 M. An apparent K _m of less than 1 M can be inferred. Uptake rates were independent of N-source during growth, and of assay pH over the range 6.2–8.7. Bicarbonate was needed for uptake, but the rate of uptake was not influenced by the simultaneous presence of NaNO₃ (10 mM) or CH₃NH₃Cl (0.15 mM). Uptake was energydependent, and was eliminated in dark, anaerobic conditions or by the addition of protonophores. Uptake was also strongly inhibited by dicyclohexylearbodiimide, an ATPase inhibitor, by — SH reagents and methionine sulfoximine, suggesting that interference with energy supply or with ammonia metabolism prevented further entry into the cells.Non-standard abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU dichlorophenyl dimethylurea - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoate - MSX L-methionine Dl-sulfoximine     

Etoposide differentially affects bone marrow and dermal derived endothelial cells

Chemotherapy alteration of the bone marrow microenvironment has the potential to influence hematopoietic recovery following transplantation. To discern the effect of specific drugs on components of the complex marrow microenvironment, in vitro models have significant utility. In the current study we sought to determine whether dermal (HMEC-1) and marrow derived endothelial cells (BMEC-1) respond differently to identical chemotherapy exposure. BMEC-1 cells were consistently more sensitive to etoposide exposure than HMEC-1 cells, measured as reduced viability. BMEC-1 also had reduced focal adhesion kinase (FAK) and VCAM-1 protein expression following chemotherapy, in contrast to dermal derived endothelial cells in which neither protein was influenced dramatically by etoposide. The two endothelial cell lines had markedly different levels of baseline VE-Cadherin protein, which was modestly altered by treatment. These data indicate that marrow derived endothelial cells have disruption of specific proteins following chemotherapy that may influence their ability to facilitate hematopoietic cell entry or egress from the marrow. In addition, these observations suggest that while BMEC-1 and HMEC-1 share a variety of characteristics, they differ significantly in their response to stress and should be incorporated into specific models with this consideration.     

Cell motility and SCAR localisation in axenically growing Dictyostelium cells

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.