David A. Cotter

High cAMP in spores of Dictyostelium discoideum : association with spore dormancy and inhibition of germination

Signalling mechanisms involving cAMP have a well-documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. The involvement of cAMP in the poorly understood developmental stages of spore dormancy and germination have been investigated in this study. Dormant spores contained up to 11-fold more cAMP than nascent amoebae. The spore cAMP levels were not constant, but typically underwent a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high cAMP levels decreased only during successful spore germination, i.e. emergence of nascent amoebae. The temporal pattern of cAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore cAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high cAMP level, and spore cAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, cAMP concentrations rose and ultimately levelled off at the high levels typical of dormant spores. The correlation between high cAMP and failure to germinate was also evident when autoactivation was inhibited by the cAMP analogue, 8-bromo-cAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued cAMP signalling in spores up to 18 d after sporulation and for linkages between elevated cAMP, spore deactivation and inhibition of spontaneous germination.

Ammonium phosphate in sori of Dictyostelium discoideum promotes spore dormancy through stimulation of the osmosensor ACG

The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.

Endogenous Autoinhibitors Regulate Changes in Actin Tyrosine Phosphorylation During Dictyostelium Spore Germination

Phosphorylation of proteins on tyrosine residues has been shown to govern many cellular processes, but little work has focused on the role of tyrosine phosphorylation during germination. Under optimal conditions, D. discoideum spores synchronously germinate each liberating a single amoeba. The total amount of phosphotyrosine containing proteins observed in spores was greatest during quiescence with a gradual decline during spore activation and emergence of nascent amoeba. During dormancy, tyrosine residues of actin were heavily phosphorylated, but they gradually underwent dephosphorylation upon spore activation and this process continued through emergence. Interestingly, an endogenous autoinhibitor(s), which blocks germination, induces tyrosine phosphorylation of actin. Conversely, the removal of the autoinhibitor(s) was followed by a decrease in phosphorylation. Thus, during germination of Dictyostelium spores, actin is dephosphorylated, with the level of phosphorylation regulated by the autoinhibitor(s) and/or the autoactivator. This change in actin phosphorylation appears to play a direct role since actin dephosphorylation and reorganization is a necessary prelude to germination.

Transcriptional Transitions during Dictyostelium Spore Germination

ABSTRACT Many protozoa form spores in response to adversity; therefore, spore germination is a key process in their life cycle. Dictyostelium discoideum sporulates in response to starvation following a developmental program. Germination is characterized by two visible changes, spore swelling and the emergence of amoeba from the spore capsule. Several studies have indicated that an additional process termed spore activation is also required, but the physiological changes that characterize the three phases are largely uncharacterized. We used microarrays to monitor global transcriptional transitions as a surrogate measure of the physiological changes that occur during germination. Using two independent methods to induce germination, we identified changes in mRNA levels that characterized the germination process rather than changes that resulted from the induction method. We found that germination is characterized by three transitions. The first transition occurs during activation, while the spores appear dormant, the largest transition occurs when swelling begins, and the third transition occurs when emergence begins. These findings indicate that activation and swelling are not passive occurrences, such as dilution of inhibitors or spore rehydration, but are active processes that are accompanied by dramatic events in mRNA degradation and de novo transcription. These findings confirm and extend earlier reports that genes such as celA are regulated during spore germination. We also found by mutation analysis that the unconventional myosin gene myoI, which is induced during early germination, plays roles in the maintenance of dormancy and in spore swelling. This finding suggests that some of the observed transcriptional changes are required for spore germination.

The MADS-box transcription factor SrfA is required for actin cytoskeleton organization and spore coat stability during Dictyostelium sporulation

The MADS-box transcription factor SrfA is involved in spore differentiation in Dictyostelium [Development 125 (1998) 3801]. Mutant spores show an altered morphology and loss of viability. A detailed structural analysis of mutant spores has been performed to gain insight into the specific aspects of spore differentiation in which SrfA is involved. Two main structural defects have been observed. One is the formation of high order actin structures, the so-called actin rods. SrfA mutant spores showed the initial stages of rod formation but no mature rods were found in older spores either in the nucleus or the cytoplasm. Moreover, phosphorylation of actin, that is believed to stabilize the actin rods, is strongly reduced in the mutant. The other defect observed was the formation of the spore coat. Young srfA- spores show basically normal trilaminar coat structures suggesting that release of prespore vesicles and basic assembly of the coat takes place in the absence of SrfA. However, the outer layer gets wavier as the spore ages and suffers a progressive degradation suggesting a late defect in the stability of the spore coat. Taken together, these results suggest that SrfA is involved in late events of spore maturation necessary for spore stability.

Vacuolar (Lysosomal) Trehalase of Saccharomyces cerevisiae

In the yeastSaccharomyces cerevisiae thePEP4 gene product, protease A, is responsible for activating all soluble vacuolar (lysosomal) enzymes. These vacuolar enzymes remain inactive inpep4 mutants. Vacuolar trehalase activity was diminished in such mutants as well. This suggests that the vacuolar (lysosomal) trehalase is processed in a manner similar to other vacuolar enzymes inS. cerevisiae.

The characterization and secretion pattern of the lysosomal trehalases ofDictyostelium discoideum

Abstract The enzyme trehalase II of Dictyostelium discoideum is efficiently secreted into the matrix of sori along with seven known lysosomal enzymes. The vegetative form of the enzyme, trehalase I, is particulate but the enzyme is secreted prior to cell aggregation or when cells are starved in phosphate buffer under standard secretion conditions. The secreted enzyme possesses properties common to lysosomal enzymes. Polyclonal and monoclonal antibodies raised against purified lysosomal N -acetylglucosaminidase precipitate the enzyme. The enzyme is released efficiently and about 62% of the initial cellular enzyme becomes extracellular. The secretion of trehalase is slightly sensitive to cycloheximide and completely blocked by sodium azide. Secretion is enhanced in the presence of disaccharides such as sucrose, lactose, and trehalose. Electrophoretograms of intracellular and secreted enzyme reveal no major processing of the enzyme during secretion. The p I of the trehalases has been estimated to be less than 2.5.

Utilization of trehalose during Dictyostelium discoideum spore germination.

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.

Dictyostelium discoideum spore germination : increases in proteinase activity are not directly coupled to the emergence of myxamoebae

SUMMARY: The accumulation of proteinase activity during the germination of Dictyostelium discoideum spores was examined under conditions in which the timing of germination events was varied. Spores had a single major proteinase, the aspartic proteinase ddAP58 (proteinase E), while the extracellular matrix which surrounds the spores in the fruiting body contained a number of lower-Mr cysteine proteinases, the most active being ddCP18. Very little peptide-nitroanilide-hydrolysing activity was detectable in spores, although some was associated with the matrix. During spore germination there were large increases in activity towards N-carbobenzoxy-L-tyrosyl-L-lysyl-L-arginine 4-nitroanilide and N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide. The enzymes responsible for these activities (referred to as ZYKRase and BzPFRase respectively) were not identical as BzPFRase was much more sensitive to the cysteine proteinase inhibitor E-64 than was ZYKRase. When spores were heatactivated, the increases in activity coincided with the emergence of myxamoebae and the appearance of cysteine proteinases detected using electrophoresis in gelatin gels. For autoactivated spores, emergence was delayed by 0·5 to 1 h and proteinase accumulation lagged slightly behind emergence. If the spores were activated with DMSO, or if heat-activated spores were treated with sucrose after swelling, proteinase accumulation proceeded more rapidly than emergence. Thus temporal control of the accumulation of proteinases during germination varies according to the conditions used. In heat-activated spores, the timing of the increase was similar to that observed previously for β-glucosidase and trehalase, but the temporal controls were not the same as those for the latter enzymes when the other activation conditions were used. The results show that proteinase accumulation is not directly coupled to emergence but could be more closely linked to the late swelling stage which precedes emergence.

The Dictyostelium discoideum prespore-specific catalase B functions to control late development and to protect spore viability

Changes in the levels of reactive oxygen species (ROS) have been associated previously with cell differentiation and development in several systems. Thus, there is interest in studying the developmental regulation of antioxidant enzymes, whose activities may modulate ROS levels and subsequent oxidant-mediated signal transduction events in specific tissues. Our recent identification in Dictyostelium discoideum of the prespore-specific catalase B (CatB) enzyme suggested (a) that the CatB enzyme functions to provide protection to the mature spores, and (b) that the CatB enzyme may have a regulatory role in cell differentiation and morphogenesis. We have now confirmed both these hypotheses. We specifically disrupted the catB gene by homologous recombination. The resulting catB null strain displays a 4-h delay in development at the time of normal catB gene expression, followed by slow and asynchronous development of fruiting bodies, taking 10 h longer than the isogenic parent strain. The expression of both prestalk- and prespore-specific genes was altered in the mutant both temporally and quantitatively, and the resultant mutant spores had increased sensitivity to H(2)O(2). This study supports the idea that CatB functions in the development of D. discoideum by regulating the level of ROS, and adds to the growing body of evidence for regulatory roles for ROS.