The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.     

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway.

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.     

Primary structure and regulation of vegetative specific genes of Dictyostelium discoideum

We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 hours. In contrast, when cells were grown in axenic broth, the mRNA levels remained constant until a dramatic drop occurred around 10 to 12 hours. Thus, regulation of both classes of genes during the first several hours of development is dependent upon the prior growth conditions. Analysis of genomic clones has resulted in the identification of two V genes, V1 and V18, as ribosomal protein genes. Several other V genes were not found to be ribosomal protein genes, suggesting that in Dictyostelium non-ribosomal protein genes may be coordinately regulated with the ribosomal protein genes. Finally, using deletion analysis we show that the promoters of two of the V genes are composed of a constitutive positive element(s) located upstream of sequences involved in the regulated expression of these genes and within the first 545 upstream bp for V18 and 850 bp for V14. The regions involved in regulated expression were localized between -7 and -222 for V18 and -70 and -368 for V14. The sequences conferring protein synthesis sensitivity were shown to reside between -502 and -61 of the H4 promoter.     

The Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.     

Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Different molecular mechanisms for cAMP regulation of gene expression during Dictyostelium development

Alterations in cAMP concentrations have been implicated in developmentally regulated gene expression in Dictyostelium. Using a variety of culture conditions to control the metabolism of cAMP during cytodifferentiation, I have examined the role of the cyclic nucleotide in development. Conditions which allow intracellular synthesis of cAMP promote the normal developmental repression of gene M4-1 by a mechanism which is completely independent of the formation of multicellular aggregates. If, however, cells are inhibited in their ability to activate adenylate cyclase and, thus, intracellular cAMP signaling, they prove unable to repress M4-1, even in the presence of exogenous cAMP. In contrast, expression of genes which exhibit maximal activity after aggregate formation depends upon accumulation of extracellular cAMP. Inhibition of intracellular cAMP signaling does not prevent the expression of these genes if cultures are simultaneously exposed to high levels of exogenously added extracellular cAMP. These results indicate that there are at least two independent mechanisms involved in the developmental regulation of gene expression by cAMP in Dictyostelium. I discuss plausible molecular mechanisms through which cAMP might alter gene expression.     

Regulation of protein synthesis during the preaggregative period of Dictyostelium discoideum development: involvement of close cell associations and cAMP

The preaggregative period of Dictyostelium discoideum is composed of two rate-limiting components which exhibit dramatic differences in either their dependency upon, or sensitivity to, close cell-cell associations, inhibitors of protein synthesis, temperature, and pH. The first component comprises the initial 4.5 hr and the second component the last 2.5 hr of the preaggregative period. By pulse-labeling cells with [35S]methionine, separating polypeptides by 2D-PAGE, and semiquantitatively comparing the rates of synthesis of 778 individual polypeptides by fluorography, the following results were obtained: a detailed program of protein synthesis accompanies the preaggregative (0-7 hr) and aggregative (7-10.5 hr) periods of development; this includes significant decreases in the rate of synthesis of 93 polypeptides synthesized during vegetative growth and significant increases in the rate of synthesis of 74 polypeptides either undetectable or synthesized at relatively low rates during vegetative growth; 35 polypeptides are transiently synthesized at different times during the preaggregative and aggregative periods; two peaks of activity are clearly defined for both increases and decreases; these peaks correlate temporally with the first and second rate-limiting components of the preaggregative period; the majority of changes (74%) which occur during the first rate-limiting component will occur in the absence of close cell-cell associations, but the majority (66%) which normally occur during the second rate-limiting component do not occur in the absence of close cell-cell associations; a high concentration of cAMP in the medium of continuous suspension cultures does not stimulate most of the changes which are dependent upon close cell-cell associations; even though cAMP stimulates progress through the second rate-limiting component in suspension cultures first allowed to associate for 4.5 hr ("competent" cells) prior to disaggregation it still does not stimulate most of the changes which are dependent upon close cell-cell associations; and synthesis of only 3 out of 778 polypeptides appears to be stimulated by addition of exogenous cAMP, and only in resuspended cultures of "competent" cells. The prominent role of close cell-cell association and the surprisingly minor effect of cAMP in the regulation of the program of protein synthesis accompanying the preaggregative and aggregative periods of Dictyostelium are discussed, especially as they relate to the effect of cAMP on protein synthesis in suspended cultures of postaggregative cells.     

Surface cAMP receptors mediate multiple responses during development in Dictyostelium: evidenced by antisense mutagenesis

Cell surface cAMP receptors (cARs) have been implicated in multiple aspects of development in Dictyostelium. Antisense mutagenesis has recently provided strong evidence that cARs are necessary for aggregation (Klein et al., 1988. Science (Wash. DC). 241:1467-1472). We show here that the expression of cAR1 antisense mRNA which prevents the appearance of cAR1 antigen also prevents the expression of cAMP-binding activity and blocks multiple cAMP-mediated responses. Chemotactic sensitivity to cAMP was lost as were stimulus-induced cAMP and cGMP production. Furthermore, the expression of developmentally regulated marker genes, dependent on repeated cAMP stimulation, was altered. As a result, the developmental program was severely impaired; most of the cells failed to aggregate and undergo further differentiation.