Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds

Background

Multicellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine.

Result

Adenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4 ^- and pdiA ^- ) and colony size (smlA ^- and ctnA ^- ) and restore their parental aggregate size.

Conclusion

Adenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation.     

Mating types in cellular slime molds

We have discovered mating types in three species of cellular slime mold. One of these species is $\text{Dictyostelium}\text{discoideum}$, hitherto the subject of extensive biochemical investigations, and now amenable to genetic studies.     

Negative chemotaxis in cellular slime molds.

This study confirms the suggestion of earlier workers that the vegetative amoebae of Dictyostelium repel each other while those of Polysphondylium violaceum do not. When Dictyostelium amoebae were placed in drops on thin and thick agar, the cells moved out faster on the thin agar, presumably because the repellent was more concentrated. This did not occur with Polysphondylium amoebae. Also, if 2 drops of cells were placed side by side, or a single drop was placed near an edge, in Dictyostelium there were fewer cells emerging between the drops (or near an edge) than on the far side. Polysphondylium showed no such difference. However, Polysphondylium amoebae were repelled by Dictyostelium cells (but not vice versa) when drops of each were placed beside one another. Finally, if Dictyostelium discoideum cells were placed in drops over thick and thin agar, but separated from the agar by a dialysis membrane, the cells again spread farther on the thin agar, indicating that the repellent is a dialyzable molecule.     

Regulation of cysteine proteinases during different pathways of differentiation in cellular slime molds

Cysteine proteinase activities have been determined using gelatin-SDS-PAGE analysis and assays based on peptide nitroanilides. Vegetative myxamoebae of all species examined contain high levels of cysteine proteinase activity present in multiple forms. In both Dictyostelium discoideum and Polysphondylium pallidum the proteinase content is dependent on whether the cells are grown axenically or in association with bacteria. In all instances development is accompanied by a decreased intracellular cysteine proteinase activity. This occurs during the formation of fruiting bodies in D. discoideum, microcysts in P. pallidum, and macrocysts in Dictyostelium mucoroides. Significant quantities of proteinase activity are always secreted by myxamoebae immediately on starvation. In D. mucoroides this leads to an almost total depletion of intracellular cysteine proteinases by the aggregation stage. As a consequence of this depletion it has been relatively easy to detect a developmentally regulated accumulation of cysteine proteinases at the enzyme activity level, something which has not yet proved possible with D. discoideum. Three cysteine proteinases are produced as D. mucoroides macrocysts develop and mature. In the case of microcyst formation in P. pallidum the proteinase contents of the developing cells and of the microcysts are dependent on how the myxamoebae are grown. In this developmental pathway at least, there is no absolute requirement for specific proteinases to be present (or absent) at a particular stage. The diversity of cysteine proteinases found in cellular slime molds and the variety of features apparent in their regulation suggest that they will prove to be very useful for investigating features of the structure/function relationships in this important group of enzymes.     

Behavior of cellular slime molds in the soil

Cellular slime molds are soil organisms, yet since they were discovered in 1869 they have been studied on agar surfaces. Here the behavior of a number of species is examined and it is evident that they have different responses to directional light and they all thrive in the presence of soil. While phototaxis clearly plays a significant role in their ability to come to the soil surface for dispersal, even more important are gradients in the soil: both temperature gradients known from earlier studies, and as we show here gas gradients, presumably ammonia as a repellent and oxygen as an attractant. There are numerous differences in both morphology and behavior among slime mold species, some of which are likely to be the result of natural selection to particular habitats, while others could be explained more easily by neutral phenotypic variation.     

Direct measurement of species-specific cohesion in cellular slime molds

Partially differentiated cells of two species of cellular slime molds, Dictyostelium discoideum and Dictyostelium purpureum, were labeled with isothiocyanate derivatives of fluorescent dyes. The labeled cells of each species segregated promptly when mixed and placed on moist filters. We determined whether cells studied at a time when they demonstrated this capacity to segregate showed a preferential adherence to cells of the same species. When labeled dissociated cells of each species were interacted with an unlabeled immobilized layer of cells of each species under appropriate conditions, binding was, in part, species-specific.     

两种网柄细胞状黏菌挥发性成分

采用固相微萃取气相色谱-质谱联用技术,对紫轮柄菌Polysphondylium violaceum和簇生岐柄菌Cavenderia fasciculata子实体的挥发性成分进行分析,从这两种网柄细胞状黏菌中共鉴定出32种挥发性成分,包括烃、醛、吲哚、萜、酮、酯和酚类化合物.其中,紫轮柄菌P.violaceum的挥发性成...     

Intracellular cyclic GMP-binding proteins in cellular slime molds.

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.     

网柄细胞状黏菌纯化培养基的改进

网柄细胞状黏菌传统的分离培养是在添加细菌的培养基上进行的。选取了9种网柄细胞状黏菌作为研究对象,在不饲喂任何细菌的条件下于3种培养基上进行培养,通过对生长状态的比较,发现燕麦片琼脂培养基对于纯化网柄细胞状黏菌具有较好的效果。     

网柄细胞状黏菌生态学的研究进展

网柄细胞状黏菌(简称网柄菌)是黏菌的第二大类群,对其生态特征的研究有助于深入了解这类生物适宜的生存条件和人类活动的影响。本文回顾了1869年至今的网柄菌生态学研究历史,从地理因素、植被因素、生物因素三个角度,探讨了影响网柄菌物种丰度和多度的生态因素,从而为土壤及凋落物中黏菌的物种多样性保护提供参考。     

Chemotaxis and binding of cyclic AMP in cellular slime molds

To obtain more information about how cyclic AMP mediates cell aggregation as found in some species of the cellular slime molds, we determined the maximal binding activity of cyclic AMP min different species under various environmental conditions. The binding of cyclic AMP is limited to amoebae using this cyclic nucleotide as chemotactic agent. Maximal binding activity proved to coincide with a maximal chemotactic response and to be related to the lenght of the period between the vegetative and the aggregative phase. Of the species studied, Dictyostelium discoideum has the highest cellular density of cyclic AMP receptors and is the most sensitive to cyclic AMP as attractant.At 15°C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than at 22°C. The number of cyclic AMP receptors is independent of temperature. The delay in the onset of aggregation and the increased chemotactic response in darkness is not due to a change in the maximal binding activity. The binding of cyclic AMP and its inactivation is discussed in the light of cell aggregation.