Regulation of discoidin I gene expression in dictyostelium discoideum by cell-cell contact and cAMP

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Summary We have recently reported the existence of two forms of glycogen phosphorylase (1,4--D-glucan: orthophosphate--glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 M, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt₂-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 M) for b to a conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.Abbreviations EGTA Ethyleneglycol-bis-(-aminoethyl ether) - N,N,N N-tetra acetic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

A comparison of the membrane-bound and extracellular cyclic AMP phosphodiesterases of Dictyostelium discoideum

The Dictyostelium discoideum membrane-bound and extracellular cyclic nucleotide phosphodiesterases (EC 3.1.4.17) shear several properties including the ability to react with a specific glycoprotein inhibitor and small inhibitory molecules. We have partialy purified the membrane-bound enzyme and compared its properties to those of the extracellular form. The kinetic properties of the two forms were similar except that, while associated with membrane particles, the membrane-bound form exhibited non-linear kinetics when assayed ove a broad substrate range. The isoelectric point of the membrane-bound phosphodiesterase was identical to that of the extracellular enzyme when isoelectrofocusing was done in the presence of 6 M urea. The molecular weights of membrane-bound and extracellular enzyme, determined by gel filtration, were the same following isoelectrofocusing in the presence of 6 M urea. When precipitated with an antiserum prepared against purified extracellular phosphodiesterase, the partially purified membrane-bound enzyme preparation was shown to contain a M_r 50 000 polypeptide comigrating with the extracellular enzyme during SDS polyacrylamide gel electrophoresis. When the iodinated extracellular enzyme and the iodinated M_r 50 000 polypeptide from membrane-bound enzyme were subjected to partial proteolytic digestion, similar profiles were obtained indicating extensive regions of homology.     

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。     

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.     

Cell-cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell-sorting in the multicellular structures

Previous studies have shown that the cell-cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell-cycle progression during the development of D. discoideum Ax-2 cells and its relation to the subsequent cell-sorting were analyzed in detail using synchronized cells and their pulse-labeling by 5'-bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell-cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2-phase (just before the PS-point from which cells initiate development when starved) progressed to the cell-cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2-phase (just after the PS-point) progressed through the cell-cycle after mound formation. The significance of cell-cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.     

A tissue culture analysis of the steps in limb chondrogenesis

Summary Tissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by USPHS Training Grant HD00152 from the National Institute of Child Health and Human Development, while P.B.A. was a postdoctoral trainee, and by NIH Grant HD05505 to M.S.     

Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum

To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 218 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group II, fgdC in group III, and fgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to cAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.     

Efficient Induction of Sporulation of Dictyostelium Prespore Cells by 8-Bromocyclic AMP under Both Submerged- and Shaken-Culture Conditions and Involvement of Protein Kinase(s) in Its Action

It is important to establish an experimental system in which sporulation of Dictyostelium can be induced at high cell densities to obtain sufficient amounts of materials for analysis of the molecular events leading to sporulation. 8-Bromo cAMP (Br-cAMP) was found to be effective for inducing sporulation by prespore cells of Dictyostelium discoideum NC4 at high cell densities under both submerged- and shaken-culture conditions. Ultrastructural studies revealed that the morphological changes associated with this sporulation proceeded normally in vitro. The effect of Br-cAMP was inhibited by two protein kinase inhibitors, K252a and staurosporine. Protein-phosphorylation experiments showed that Br-cAMP induced increased phosphorylations of a 96 kDa spore coat protein (SP96) and a protein with a mobility corresponding to a molecular weight of 50 kDa (p50-4). The protein kinase inhibitor K252a blocked the phosphorylations of both proteins. These proteins may be targets of particular protein kinase(s) that is activated by Br-cAMP. These findings indicate that the present experimental system should be useful for elucidating the molecular events involved in normal sporulation and the mechanism by which Br-cAMP induces sporulation in vitro.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.