Center for Molecular Genetics, Division of Biology, University of California San Diego, La Jolla, CA 92093, USA
* Present address: Dept. of Microbiology, Swedish University of Agricultural Sciences Box 7025, 750 07 Uppsala, Sweden
Author for correspondence (e-mail: wloomis{at}ucsd.edu)
Accepted June 19, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Adenylyl cyclase ACA, Adenylyl cyclase ACR, cAMP-dependent protein kinase PKA, Sporulation, Dictyostelium
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains in which acgA is disrupted develop normally into fruiting bodies but the spores fail to remain dormant when dispersed into an environment of high osmolarity (van Es et al., 1996). Strains in which acaA is disrupted do not secrete cAMP and so are unable to chemotactically signal each other. They fail to aggregate unless they are pulsed with extracellular cAMP or they are genetically engineered to overexpress the catalytic subunit of the cAMP-dependent protein kinase, PKA (Pitt et al., 1993; Wang and Kuspa, 1997). Aggregates form under these conditions only when the cells are spread at high densities and result from random collisions rather than from chemotaxis. Strains in which acrA is disrupted aggregate and proceed to make fruiting bodies but have a cell-autonomous defect in sporulation such that less than 1% of the cells make detergent-resistant viable spores (Soderbom et al., 1999). It appears that acrA cannot fulfill the role of acaA in production of the extracellular cAMP necessary for chemotaxis while acaA cannot fulfill the role of acrA in production of internal cAMP necessary for terminal differentiation of spores.
Although both acaA and acrA are expressed during development and their products catalyze the synthesis of cAMP, there are significant differences in the enzymes. ACA is similar to other G-protein coupled membrane-associated adenylyl cyclases while ACR is similar to soluble mammalian cyclases (Buck et al., 1999). ACA is activated when the G-protein coupled surface receptor CAR1 binds extracellular cAMP. In extracts of aggregation stage cells, the activity can be stimulated more than tenfold by adding GTPS to dissociate the trimeric G protein (Pitt et al., 1992). The activity can also be stimulated by addition of manganese ions which by-pass the need for G-protein-dependent activation (Loomis et al., 1978; Pitt et al., 1992). The activity of ACR in extracts, however, is not stimulated by GTP
S and is maximally active in the presence of magnesium ions (Kim et al., 1998; Meima and Schaap, 1999; Soderbom et al., 1999). During the aggregation stage, between 6 and 12 hours of development, the activity of ACA assayed in the presence of manganese is about fourfold higher than the activity of ACR assayed in the presence of magnesium (Soderbom et al., 1999). However, during culmination, ACR activity is more than tenfold higher than ACA activity. The temporal pattern of accumulation of these activities may partially account for their stage specificity. However, the adenylyl cyclases may have other differences in characteristics that dedicate them to distinct roles. Therefore, we have analyzed the effects of ectopic expression of acaA on the phenotype of cells lacking ACR and the developmental capabilities of strains lacking both of the developmental adenylyl cyclases but overexpressing PKA.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells, growth, transformation and development
Cells of strain AX4 (Knecht et al., 1986), DG1100 (acrA-) (Soderbom et al., 1999) and AK631 (acaA- PKA-Cover) (Wang and Kuspa, 1997) were grown in HL5 medium (Sussman, 1987). BlasticidinS was added to the medium at 10 µg/ml to select for cells carrying the BsrR gene. Geneticin (G418) was added to the medium at 20 µg/ml to select for cells carrying the NPT II gene. acrA was disrupted in the strain AK631 (acaA- PKA-Cover) as previously described (Soderbom et al., 1999). Strain acrA-/K was obtained by transformation of DG1100 (acrA-) with the K-Neo plasmid (pkaC::pkaC) as previously described (Anjard et al., 1992). Multiple independent transformants were isolated from each transformation and three or four were characterized.
A mutation in acaA (L394S) was selected such that its product, ACA, can function effectively in the absence of G-protein-coupled stimulation from the CAR1 receptor (Parent and Devreotes, 1995). The plasmid CP105 carrying this modified gene under control of the actin 15 regulatory region gives high levels of ACA activity even in the absence of cAMP signals. We transformed both wild-type cells and DG1100 strain cells with this construct and selected for G418 resistance using up to 50 µg/ml of G-418. The strains were called AX4 ACA* and acrA- ACA* respectively. Both strains aggregate about 2 hours earlier than wild-type cells and form smaller fruiting bodies.
Synchronous development was induced by depositing the cells on nitrocellulose filters supported on pads saturated with buffer (Sussman, 1987). For microscopic examination, structures were deposited on slides in 10-15 µl of 0.1% Calcofluor White ST in 20 mM phosphate buffer pH 6.8 and observed with a Nikon fluorescence microscope.
Quantitation of sporulation
Cells were collected off nitrocellulose filters on which they had developed for 48 hours, dissociated in buffer containing 0.5% Cemusol, incubated for 5 minutes, washed and spread on SM agar in association with Klebsiella aerogenes (Shaulsky et al., 1995). The number of plaques formed after 4 days indicated the number of viable spores.
Adenylyl cyclase assay
1x108 cells were collected at each time point, washed in 10 mM phoshate buffer pH 6.5, resuspended in 200 µl of lysis buffer (10 mM Tris pH 8.0, 250 mM sucrose). Cells were lysed through 3 µm pore size nucleopore membranes and the extracts kept on ice. The adenylyl cyclase assay was then performed as described by Soderbom et al. (Soderbom et al., 1999).
PKA assay
PKA activities were determined using the protein kinase A assay system from Gibco BRL (# 13128-012) as previously described (Anjard et al., 1993). After 20 hours of development on filters, cells were harvested in 20 mM phosphate buffer pH 6.5, centrifuged for 1 minute at 1200 rpm and resuspended in 500 µl extraction buffer containing 1/100 of protease inhibitor cocktail (Sigma P-8215). Cells were lysed by freezing in a dry ice/ethanol bath and rapidly thawing at 20°C. After centrifugation at 12,000 g for one minute, 1-10 µl from each supernatant was assayed at 25°C. The activities are expressed as phosphorylation of Kemptide substrate that can be inhibited by the PKI inhibitor, according to the instructions with the kit. The reaction was linear with extract under the conditions used. Protein concentration was measured using the Bradford assay with BSA as standard.
Northern blot
Northern blot analyses were performed as described by Shaulsky and Loomis (Shaulsky and Loomis, 1993). RNA was isolated using Triazol reagent and 15 µg RNA from each sample was electrophoretically separated on a 0.8% agarose gel and subsequently transferred to nylon membrane (Magna Graph, MSI, Westboro, MA). Probes for ecmA, cotB and pkaC mRNA were generated from appropriate plasmids.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
These results indicate that expression of ACA* under the control of the actin 15 promoter can substitute for ACR during culmination but is not sufficient for maintaining dormancy. It would also appear that ACR plays no other role essential for sporulation than catalyzing the synthesis of cAMP.
Cells lacking both adenylyl cyclase activities
It was previously shown that a strain lacking ACA but overexpressing PKA (acaA- PKA-Cover) is able to form aggregates at high cell densities by random collisions and that the aggregates transform into slugs that culminate to form fairly normal fruiting bodies (Wang and Kuspa, 1997). To determine the role of ACR in development of this strain we transformed cells of the acaA- PKA-Cover strain AK631 with a construct in which acrA was disrupted by the blasticidin resistance gene and selected for homologous recombinants. Several independent transformants in which acrA was shown to be disrupted were further analyzed. Cells of these strains were found to grow well and, when plated at high cell density, formed tipped mounds and a few small abnormal finger-like structures but no fruiting bodies (Fig. 2). No measurable adenylyl cyclase activity was found at any stage of development in these strains (data not shown).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of a modified form of the early adenylyl cyclase, ACA, that is independent of G-protein activation in acrA- null cells resulted in the formation of detergent-resistant viable spores that were able to exclude propidium iodide. The number of viable spores was highest immediately after culmination and decreased rapidly thereafter as a result of premature germination. It appears that expression of the act15::acaA* construct in acrA- cells resulted in sufficient internal cAMP for terminal differentiation of spores indicating that ACR plays no other role in sporulation than the synthesis of cAMP.
It has been proposed that Dictyostelium can develop in the absence of cAMP as long as PKA is constitutively active as a result of overexpression of the catalytic subunit from pkaC (Wang and Kuspa, 1997). While acaA- PKAover cells that lack the aggregation adenylyl cyclase are unable to chemotactically signal each other, they can form aggregates if plated at high cell density. They then proceed through subsequent stages to form fairly normal fruiting bodies complete with stalks and viable spores (Wang and Kuspa, 1997). No measurable cAMP was found to accumulate prior to culmination in these cells due to rapid turnover (Wang and Kuspa, 1997; Meima and Schaap, 1999). However, we have found that the adenylyl cyclase activity of ACR is essential for morphogenesis beyond the mound stage as well as the differentiation of viable spores. It could be argued that the act15::pkaC construct might not be sufficiently active late in development to result in constitutive PKA activity, however direct enzymatic measurements showed that PKA was constitutively active late in development of the strain lacking both developmental adenylyl cyclases but carrying the pkaC construct (Table 1). While these mutant cells accumulate mRNA from the prestalk gene ecmA to about the same level as seen in their parental strain AK631, the prespore-specific gene cotB was expressed at a very low level and almost no spores were formed. When developed in mixtures with wild-type cells, marked TL130 (acrA- acaA- PKA-Cover) cells co-aggregated and were found in both the anterior prestalk region and the posterior prespore region suggesting that they had sorted out normally. Although normal fruiting bodies were subsequently formed, the mutant cells failed to give rise to viable spores further confirming the cell autonomous nature of the acrA- defect in sporulation.
While cells of strain TL130 are blocked at the tipped mound stage of morphogenesis when developed as pure populations, they can proceed to form fruiting bodies complete with stalks and sori when developed together with 10% wild-type cells. Although the chimeric fruiting bodies are fairly small, it is unlikely that the stalks are formed only from wild-type cells since they make up only a minor proportion of the total cells. It is more likely that factors secreted by wild-type cells direct the morphological progression from the finger stage to culmination and induce stalk differentiation among the acrA- mutant cells. The signaling factor secreted by wild-type cells is unlikely to be cAMP since neither 5 mM cAMP nor 500 µM 2' deoxy-cAMP added to 16-hour-developed TL130 cells led to fruiting body formation (unpublished observations).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anjard, C., Pinaud, S., Kay, R. R. and Reymond, C. D. (1992). Overexpression of DdPK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum. Development 115, 785-790.
Anjard, C., Etchebehere, L., Pinaud, S., Veron, M. and Reymond, C. D. (1993). An unusual catalytic subunit for the cAMP-dependent protein kinase of Dictyostelium discoideum. Biochem. 32, 9532-9538.[Medline]
Buck, J., Sinclais, M., Schapal, L., Cann, M. and Levin, L. (1999). Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA 96, 79-84.
Ceccarelli, A., Mahbubani, H. and Williams, J. G. (1991). Positively and negatively acting signals regulating stalk cell and anterior-like cell differentiation in Dictyostelium. Cell 65, 983-989.[Medline]
Kim, H. J., Chang, W. T., Meima, M., Gross, J. D. and Schaap, P. (1998). A novel adenylyl cyclase detected in rapidly developing mutants of Dictyostelium. J. Biol. Chem. 273, 30859-30862.
Knecht, D. A., Cohen, S. M., Loomis, W. F. and Lodish, H. F. (1986). Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Mol. Cell. Biol. 6, 3973-3983.[Medline]
Loomis, W. F., Klein, C. and Brachet, P. (1978). The effect of divalent cations on aggregation of Dictyostelium discoideum. Differentiation 12, 83-89.[Medline]
Meima, M. E. and Schaap, P. (1999). Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation. Dev. Biol. 212, 182-190.[Medline]
Parent, C. A. and Devreotes, P. N. (1995). Isolation of inactive and G protein-resistant adenylyl cyclase mutants using random mutagenesis. J. Biol. Chem. 270, 22693-22696.
Pitt, G. S., Brandt, R., Lin, K. C., Devreotes, P. N. and Schaap, P. (1993). Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA). Genes Dev. 7, 2172-2180.[Abstract]
Pitt, G. S., Milona, N., Borleis, J., Lin, K. C., Reed, R. R. and Devreotes, P. N. (1992). Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. Cell 69, 305-315.[Medline]
Shaulsky, G., Kuspa, A. and Loomis, W. F. (1995). A multidrug resistance transporter serine protease gene is required for prestalk specialization in Dictyostelium. Genes Dev. 9, 1111-1122.[Abstract]
Shaulsky, G. and Loomis, W. F. (1993). Cell type regulation in response to expression of ricin-A in Dictyostelium. Dev. Biol. 160, 85-98.[Medline]
Shaulsky, G. and Loomis, W. F. (1996). Initial cell type divergence in Dictyostelium is independent of DIF-1. Dev. Biol. 174, 214-220.[Medline]
Soderbom, F., Anjard, C., Iranfar, N., Fuller, D. and Loomis, W. F. (1999). An adenylyl cyclase that functions during late development of Dictyostelium. Development 126, 5463-5471.
Sussman, M. (1987). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. In Methods in Cell Biology, Vol. 28 (ed. J. A. Spudich), pp. 9-29. Orlando, FL: Academic Press.
van Es, S., Virdy, K. J., Pitt, G. S., Meima, M., Sands, T. W., Devreotes, P. N., Cotter, D. A. and Schaap, P. (1996). Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores. J. Biol. Chem. 271, 23623-23625.
Virdy, K. J., Sands, T. W., Kopko, S. H., van Es, S., Meima, M., Schaap, P. and Cotter, D. A. (1999). High cAMP in spores of Dictyostelium discoideum: association with spore dormancy and inhibition of germination. Microbiol. 145, 1883-1890.[Abstract]
Wang, B. and Kuspa, A. (1997). Dictyostelium development in the absence of cAMP. Science 277, 251-254.
Wang, N., Soderbom, F., Anjard, C., Shaulsky, G. and Loomis, W. F. (1999). SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane-spanning sensor kinase DhkA. Mol. Cell. Biol. 19, 4750-4756.