Huddling and the control of water loss by the slug Limax pseudoflavus Evans

Limax pseudoflavus Evans form closely packed huddles within their day-time resting sites. Huddles will form in two populations of this species are mixed. Huddles will also form if L. pseudoflavus and L. flavus are mixed, but individuals tend to select conspecific neighbours within the huddles. Huddles form only after considerable milling around of the component slugs and form more frequently and involve more contact in dry compared with humid conditions. Moving slugs increase their evaporation rate by 65% compared with still animals of the same weight. Two slugs making close contact reduce their evaporation rate by 34% compared with unhuddled slugs. It is argued that huddles are non-social aggregations whose prime function is the conservation of water.     

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.     

Interconnected Cavernous Structure of Bacterial Fruiting Bodies

The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.     

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.     

An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour, and culmination

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.     

A cell type-specific effect of calcium on pattern formation and differentiation in dictyostelium discoideum

Spatial gradients of sequestered and free cellular calcium (Ca2+) exist in the slug of Dictyostelium discoideum (Maeda and Maeda, 1973; Tirlapur et al., 1991; Azhar et al., 1995; Cubitt et al., 1995). When we vary intracellular Ca2+ with the help of calcium buffers and the ionophore Br-A23187, there are striking effects on slug morphology, patterning and cell differentiation. In the presence of a calcium ionophore, high external Ca2+ levels lead to an increase of intracellular sequestered and free Ca2+, the formation of long slugs, a decrease in the fraction of genetically defined prespore cells and 'stalky' fruiting bodies. Conversely, a lowering of external Ca2+ levels results in a decrease of intracellular Ca2+, the formation of short slugs, an increase in the prespore fraction and 'spory' fruiting bodies. We infer that Ca2+ plays a significant morphogenetic role in D. discoideum development, by selectively promoting the prestalk pathway relative to the prespore pathway.     

WATER-REGULATORY BEHAVIOUR IN TERRESTRIAL GASTROPODS

1. Terrestrial snails and slugs are exceedingly susceptible to dehydration due to evaporative water loss from the integument and lung surface, and the deposition of a dilute mucous trail. Active slugs can lose 30–40% of their initial body weight (IBW) within 2 h. 2. Both field and laboratory studies have revealed that habitat selection by snails and slugs is well correlated with the availability of water. In addition, numerous species display homing behaviour, returning directly to their moist secluded daytime resting sites at dawn. 3. Several aspects of locomotor activity are affected by body hydration and environmental conditions such as relative humidity. Moist conditions result in termination of aestivation in snails and a generally higher level of activity in both snails and slugs. In contrast, severe dehydration initiates aestivation in snails and an increase in the intensity and duration of circadian locomotor activity in slugs. 4. Huddling behaviour is a specialized example of the general preference of slugs for moist habitats. When groups of slugs are exposed to dry environmental conditions, they form closely packed aggregations. This response results in a decrease in the rate of dehydration of the individual slugs. 5. When slugs have been dehydrated to about 90% IBW, rhythmic closures of the pneumostome are initiated. As dehydration progresses there is a reduction in the open diameter of the pneumostome. These responses reduce the total exposure of the lung surface and thereby evaporative water loss. In slugs dehydrated to about 80 % IBW, these responses can result in a 7 % reduction in water loss. 6. When slugs have been dehydrated to about 65% IBW (67·6 ± 4·3% IBW) they move on to a moist surface, assume a characteristic flattened posture and remain quiescent while water is absorbed through the surface of the foot. Once they are rehydrated (to 93·5 ± 12·4% IBW) they move off the moist surface. Thus there exists a specific dehydration threshold for the initiation of contact-rehydration and a rehydration set-point at which the response is terminated. 7. Both initiation and termination of contact-rehydration are controlled by variations in haemolymph osmotic pressure. The behaviour can be experimentally initiated by injection of hyperosmotic mannitol solution and terminated by injection of dilute saline. 8. Contact-rehydration involves bulk flow of water through an epithelial paracellular pathway in the integument of the foot. The rate of absorption of [¹⁴C]inulin during contact-rehydration is similar to that of water. The efficacy of water movement through the pathway is modulated by body hydration, the pathway being opened only in dehydrated slugs. 9. By means of the dual-limit control of contact-rehydration slugs can behaviourally regulate their body hydration and haemolymph osmolality within the tolerable hydration range described by the upper and lower limits.     

Exploiting new terrain: an advantage to sociality in the slime mold Dictyostelium discoideum

Understanding the ecological benefits of social actions is centralto explaining the evolution of social behavior. The social amoebaDictyostelium discoideum has been well studied and is a modelfor social evolution and development, but surprisingly littleis known about its ecology. When starving, thousands of thenormally solitary amoebae aggregate to form a differentiatedmulticellular organism known as a slug. The slug migrates towardthe soil surface where it metamorphoses into a fruiting bodyof hardy spores held up by a dead stalk comprising about one-fifthof the cells. Multicellularity in D. discoideum is thought tohave evolved to lift the spores above the hazards of the soilwhere spores can be picked up for long-distance dispersal. Here,we show that multicellularity has another advantage: local dispersalto new food sources. We find that cells shed by D. discoideumslugs during migration consume and remove bacteria in the pathof the slug, although slugs themselves do not breakup. We alsoshow that slugs are adept at local dispersal by comparing migrationof slugs with migration of individual cells of the mutant, CAP2,which cannot aggregate and so rely only on cellular movement.In particular, the solitary cells of the aggregation mutantare unable to cross a soil barrier, easily crossed by slugs.We propose that the exploitation of local food patches is animportant selective benefit favoring multicellular cooperationin D. discoideum.     

Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.     

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.     

An immediate-early gene, srsA: its involvement in the starvation response that initiates differentiation of Dictyostelium cells

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a differentiation program for survival. We have found a novel gene, srsA, which is rapidly expressed in the first 5 min following the removal of nutrients and is turned off within an hour. This gene encodes a small protein with no significant similarity to previously characterized proteins. Disruption of srsA results in delayed expression of the early genes acaA and carA that encode adenylyl cyclase and the cAMP receptor necessary for chemotactic aggregation, respectively. Streaming is delayed several hours and the aggregates are larger than normal in the mutant strains. These phenotypes are cell-autonomous. Overexpression of srsA also results in delayed aggregation. Some of the slugs of the srsA(OE) strains showed stalked migration reminiscent of the slugs of the related species Dictyostelium mucoroides. The terminal structures formed by srsA(OE) cells were grossly abnormal and contained very few viable spores. When cells overexpressing srsA were developed together with an excess of wild-type cells, the fruiting bodies were still abnormal, indicating that the mutant cells have a dominant effect on late development. These findings suggest that srsA may be involved in both the starvation response and late differentiation.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

PHBHHx膜表面亲水改性及其与神经干细胞生物相容性

对羟基丁酸-羟基己酸共聚酯(PHBHHx)膜进行表面改性,研究神经干细胞(NSCs)在改性后的PHBHHx膜表面的贴附、增殖及分化情况,为开发新型脑组织工程支架材料奠定基础。采用溶剂挥发法制备PHBHHx膜,扫描电镜观察其表面性状;分别通过脂肪酶处理,NaOH处理的方法对PHBHHx膜进行表面改性,测量接触角以检测膜表面亲水性。分离培养孕14.5 d大鼠胚胎大脑皮质NSCs,接种在表面改性后的PHBHHx膜表面进行体外培养,扫描电镜观察膜表面细胞形态,MTT法检测细胞活力,免疫细胞化学染色观察NSCs存活和分化情况。结果显示,与未处理的PHBHHx膜相比,脂肪酶、NaOH处理能够显著提高PHBHHx膜表面亲水性,增加NSCs在PHBHHx膜表面贴附数量;NSCs在改性后的PHBHHx膜表面能够良好地存活并分化为神经元和胶质细胞。结果提示PHBHHx膜表面碱处理通过提高材料表面亲水性和粗糙程度,增加其与NSCs的生物相容性,改性后的PHBHHx材料是一种非常有潜力的新型脑组织工程支架材料,有望在NSCs移植修复脑损伤中发挥作用。     

Role of cyclic AMP and ammonia in induction and maintenance of post-aggregative differentiation in a suspension culture of Dictyostelium discoideum

The effects of ammonia and cAMP on prespore and prestalk differentiation of Dictyostelium discoideum were investigated by monitoring eight developmentally regulated proteins as differentiation markers under the shake culture conditions in glucose/albumin medium. In the medium containing cAMP, cells form small agglomerates and undergo prespore differentiation [19]. Under the conditions where agglomeration was prevented, ammonia induced four marker proteins out of eight tested in the presence of cAMP, which included not only a prespore specific enzyme but also cell-type non-specific proteins. No inhibitory effect of ammonia was observed in presumptive cell differentiation. These results suggest that ammonia is an inducer of differentiation at the protein level as well as the mRNA level as found previously [24]. The effects of cAMP were examined with special attention to the difference between induction of differentiation and maintenance of differentiated state in this specific medium. The induction of differentiation from early aggregative cells was cAMP-dependent with all the marker proteins tested. This agrees with the observations so far obtained in other culture systems. However, when already differentiated cell masses (slugs) were dissociated and shaken in this specific medium, only two enzymes required cAMP to maintain the activity while five out of eight kinds of the proteins continued to be expressed as in undisturbed slugs even without cAMP. This suggests that for the maintenance of the differentiated state after slug disaggregation cAMP may not be required with respect to the majority of proteins, if cells are provided with some favorable conditions such as glucose/albumin medium.     

A putative Ariadne-like ubiquitin ligase is required for Dictyostelium discoideum development

The Dictyostelium rbrA gene encodes a putative Ariadne ubiquitin ligase. rbrA⁻ cells form defective slugs that cannot phototax. Prestalk cell numbers are reduced in rbrA⁻ slugs, and these prestalk cells do not localize to the tip of slugs. Chimeric slugs containing wild-type cells could phototax and form fruiting bodies.     

THE ACTION OF METALDEHYDE ON THE SLUG AGRIOLIMAX RETICULATUS (MÜLLER)

Metaldehyde both as powder and in solution can act on slugs either by contact or as a 'stomach poison'. The characteristic effects of metaldehyde poisoning were immobilization broken by outbursts of unco-ordinated muscular activity and sliming which usually resulted in severe water loss. 24 hr. after treatment with moderate doses slugs were still abnormal and rarely fed within 30 hr. of treatment. It was not possible to determine the M.L.D. with the methods used, but 0·06 nig. solid metaldehyde taken orally could be lethal to slugs of 400–800 mg. body weight. Lethal effects were produced by contact of 1 hr. with concentrations equivalent to 0·0063 mg./cm.². Toxicity increased with rise in temperature and recovery from moderate doses was dependent on slugs being in a saturated or almost saturated atmosphere. No obvious gut lesions were found in slugs which had been dosed with or had eaten metaldehyde. Its action was not by depolymerization in the gut or body cavity. In the light of laboratory and small scale field trials it is suggested that broadcasting and spraying are the best methods of applying the material.     

How a fungus escapes the water to grow into the air

Fungi are well known to the casual observer for producing water-repelling aerial moulds and elaborate fruiting bodies such as mushrooms and polypores. Filamentous fungi colonize moist substrates (such as wood) and have to breach the water-air interface to grow into the air. Animals and plants breach this interface by mechanical force. Here, we show that a filamentous fungus such as Schizophyllum commune first has to reduce the water surface tension before its hyphae can escape the aqueous phase to form aerial structures such as aerial hyphae or fruiting bodies. The large drop in surface tension (from 72 to 24 mJ m-2) results from self-assembly of a secreted hydrophobin (SC3) into a stable amphipathic protein film at the water-air interface. Other, but not all, surface-active molecules (that is, other class I hydrophobins and streptofactin from Streptomyces tendae) can substitute for SC3 in the medium. This demonstrates that hydrophobins not only have a function at the hyphal surface but also at the medium-air interface, which explains why fungi secrete large amounts of hydrophobin into their aqueous surroundings.     

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.