Present address: Department of Biology, University of California, San Diego, LaJolla, CA 92093-0568, USA
Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA
* These authors contributed equally to this study
Author for correspondence (e-mail: alexanderh{at}missouri.edu)
Accepted June 20, 2001
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SUMMARY |
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Key words: Cisplatin resistance, Sphingolipids, Cell motility, Morphogenesis, Gene expression, Actin, Pseudopodia, EDG receptors, G proteins, GPI anchor, Dictyostelium discoideum
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INTRODUCTION |
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There is growing support for an intracellular second messenger role for S-1-P as well. Microinjection of Swiss 3T3 cells with S-1-P induces DNA synthesis (Van Brocklyn et al., 1998), and the sphingosine kinase inhibitor D,L-threo-dihydrosphingosine blocks platelet derived growth factor stimulated S-1-P accumulation and ERK activation (Rani et al., 1997; Tolan et al., 1997). More importantly, overexpression of the S-1-P kinase in NIH 3T3 cells results in an increase in S-1-P, and a concomitant increase in DNA synthesis, cell growth and cell number. In addition, overexpression of S-1-P kinase protects the cells from ceramide-induced cell death. Importantly, no S-1-P was detected in the medium, indicating that S-1-P was functioning intracellularly (Kohama et al., 1998). Although the precise underlying mechanisms remain elusive, it is evident that the enzymes that modulate the concentration of these bioactive sphingolipids are important regulatory elements, controlling cell fate decisions and development.
Dictyostelium discoideum is an attractive organism to use for the study of cell and developmental biology, and the complex program of gene expression that underlies and controls cell-type differentiation and morphogenesis in this organism is becoming increasingly well understood (Kessin, 2001). Cells divide mitotically and remain as single cells as long as there is adequate food. When the food is depleted, the cells initiate the developmental program and groups of 1x105 cells aggregate chemotactically to form multicellular structures. These aggregates then proceed synchronously through the morphogenetic program, culminating in the construction of fruiting bodies, each with a mass of 80,000 spores, resting atop a slender multicellular stalk. The roles of cAMP, G-protein-coupled receptors and phosphoinositides in controlling cell motility, chemotaxis and gene expression have been intensively investigated (Laurence and Firtel, 1999; Parent and Devreotes, 1999).
We recently used the REMI (restriction enzyme mediated integration) insertional mutagenesis technique to identify Dictyostelium mutants with increased resistance to the widely used anticancer drug cisplatin (Li et al., 2000). One of the resistant mutants identified in the study possessed a disrupted sglA gene, which encodes S-1-P lyase. The S-1-P lyase enzyme acts at the last step in sphingomyelin catabolism, and is responsible for the degradation of S-1-P to phosphoethanolamine and hexadecanal (Fig. 1) (van Veldhoven and Mannaerts, 1993). This was the first identification of a component of this pathway in Dictyostelium, as well as the first implication of the involvement of this enzyme in cisplatin resistance.
In addition to resistance to cisplatin, the S-1-P lyase null mutant had a strong developmental phenotype and produced aberrant fruiting bodies, with short thick stalks and no obvious apical spore mass (Li et al., 2000). Previously, the S-1-P lyase gene had only been deleted in the yeast Saccharomyces cerevisiae (Gottlieb et al., 1999). Using the Dictyostelium S-1-P lyase null mutant, we have been able to examine the role of this enzyme in multicellular development. Our results show that S-1-P lyase functions at multiple steps throughout the course of development, including the cytoskeletal architecture of aggregating cells, slug migration, developmental gene expression and spore differentiation. In addition, cells that lack S-1-P lyase show increased viability in stationary phase. This pleiotropic effect, which is due to the loss of S-1-P lyase, establishes sphingolipids as central regulatory molecules in Dictyostelium growth and development.
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MATERIALS AND METHODS |
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Homologous disruption of the sglA gene
An 831 bp fragment of sglA was amplified from genomic DNA by PCR, using the 5' primer, 5'-CTAGTCTAGAGTTTCCC-ATCAATTCG (#225) and the 3' primer, 5'-CAAGAGAATGC-AACAACC (#226). The amplified DNA fragment was digested with XbaI and XhoI and cloned into XbaI-XhoI digested pBluescript KS(+) to generate plasmid pCis2B. A 1.4 kb BamHI cassette, containing the blasticidin S resistance (bsr) gene (Kamakura et al., 1987), was purified from plasmid SL63 (obtained from R. Firtel, University of California, San Diego, CA) and inserted into the BamHI site within the sglA-coding region, to generate plasmid Rcis2B, to be used for gene disruption. An XbaI-XhoI fragment from Rcis2B containing the bsr gene was used for disruption of the endogenous sglA gene.
DNA transformation and selection were performed as described (Kuspa and Loomis, 1992). Briefly, 1.2x107 Ax4 cells were transformed with 20 µg of excised DNA fragment at 1kV, 3 µF in 4 mm-gap-width cuvettes using the BioRad Gene pulser. Cells were cured with 1 mM CaCl2 and 1 mM MgCl2 for 10 minutes, and then were resuspended in 40 ml HL5 medium and transferred to petri dishes. After 24 hours, 10 µg/ml blasticidin S (ICN, Costa Mesa, CA) was added and the plates were incubated at 22°C. Colonies began to appear after 7-10 days. To isolate single clones, transformed cells were plated on SM agar plates with Klebsiella aerogenes (Sussman, 1987).
All standard DNA manipulations were performed according to published methods (Sambrook et al., 1989). Genomic DNA was prepared using DNAzol reagent, and total RNA was isolated using TRI reagent (both from Molecular Research Center, Cincinnati, OH). PCR fragments were purified using GENECLEAN III (BIO 101, Vista, CA).
Southern and northern analyses
Southern and Northern analyses were performed as described (Lee et al., 1997). DNA (20 µg/lane) or total RNA (10 µg/lane) were used for Southern and northern analyses, respectively. Equal loading of RNA was monitored by staining for rRNA with 0.25 µg/ml Acridine Orange. Northern blots were quantified by using a FUJIFILM FLA-2000 PhosphorImager. Southern nylon blots were stripped by pouring a boiling solution of 0.1xSSC (15 mM NaCl, 1.5 mM Na3C6H5O7·2H2O, pH 7.0) containing 0.1% SDS over the blot in a Pyrex dish. The blots were shaken slowly for 15 minutes at room temperature, and the procedure was repeated three times. To test for complete removal of the labeled probe, the blots were re-scanned using the PhosphorImager. The following hybridization probes were used: sglA, a 0.83 kb PCR product from genomic DNA; bsr, a 1.4 kb BamHI fragment from SL63; CSA, c512 plasmid, linearized with BglII; ecmB, a 2.4 kb HindIII fragment from pDd56; PsA, a D19 plasmid, linearized with NsiI; catBl, a 2.1 kb SalI-NotI fragment from SLK452; and spiA, a 0.45 kb PCR product amplified from genomic DNA.
Strains and conditions for growth and development
Ax4 was the parental strain used for the generation of the sglA mutant by direct homologous recombination. Cells were grown in HL-5 medium and passed, or used for experiments, when the cell density reached 2-3x106 cells/ml (Cocucci and Sussman, 1970). New cultures were started monthly from stored spores. Cell numbers was monitored by counting in a hemacytometer. For development, cells were harvested and washed in LPS buffer (20 mM KCl, 2.5 mM MgCl2, 40 mM K-PO4, pH 6.5 containing 0.5 mg/ml streptomycin sulfate). Aliquots of 1x108 washed cells were deposited on 40 mm diameter black paper filters (Thomas Scientific) supported by LPS buffer-saturated Gelman cellulose filter pads, and were allowed to develop at 22°C (Soll, 1987; Sussman, 1987). The cells on each filter aggregate to form approximately 1000 multicellular assemblies, which proceeded through the remainder of development in synchrony. Photographs were taken at hourly intervals to record morphogenetic changes. Developing cells were harvested by vortexing the filters in 2 ml SS buffer (10 mM NaCl, 10 mM KCl, 2.7 mM CaCl2). The samples were either used directly for experiments or frozen as pellets for later molecular analyses.
Immunofluorescence staining
Cells were allowed to develop on black paper filters until the onset of aggregation at 5 hours. The aggregating cells were harvested, disaggregated by vortexing and washed twice in LPS buffer. The washed cells were deposited at 2x104 cells/cm2 on ethanol-washed coverslips. After 10 minutes, the coverslips were flooded with a fixative solution of 3.7% formaldehyde in LPS, and incubated for 5 minutes at room temperature. To permeabilize the cells, the fixative solution was replaced with a solution of 0.5% NP-40 in LPS, and incubated for 5 additional minutes. The coverslips were then washed three times with LPS, and incubated overnight in 1% bovine serum albumin (BSA) in LPS at 4°C (Alexander et al., 1992). For F-actin staining, cells were incubated for 20 minutes with 25 nM rhodamine-labeled phalloidin in 1% BSA/LPS. Coverslips were then washed three times with LPS and mounted in a solution of Airvol (Air products, Allentown, Pa., USA) containing 50 mg/ml DABCO (1,4-diazabicyclo[2-2-2]octane) to prevent fading of fluorescence. Slides from four different experiments were examined and photographed with a Zeiss IM inverted microscope using a 100x Neofluor lens.
Slug migration and phototaxis assay
Cells were grown axenically to a density of 2-3x106 cells/ml, harvested by centrifugation, washed twice with water and suspended in water to a final concentration of 5x108 cell/ml. 0.1 ml of this suspension was deposited as a 3 cm line at one end of a 1.5% H2O-agar plate. The plates were wrapped in foil, a pin hole was made in the foil at the end opposite to where the cells were deposited and the plates were incubated at 22°C in a lighted incubator (Newell et al., 1969). After two days, the plates were removed and the slugs and slime trails were lifted onto 0.2 mm thick polyvinylchloride discs, which were then air dried, stained for 30 seconds with 0.3% (w/v) Coomassie Blue in 50% ethanol/10% acetic acid, washed and blotted dry for a permanent record of the experiment (Fisher et al., 1981).
Sporulation efficiency assay
Mutants and wild-type cells were allowed to develop on black paper filters. At 24 and 48 hours, the cells were collected by vortexing the filters in 2 ml SS buffer. Spores and total cells from each sample were counted using a hemacytometer. The sporulation efficiency is defined as the percentage of the spores in the total cells counted.
S-1-P treatment
A stock solution of S-1-P was prepared by dissolving the S-1-P to 0.5 mg/ml in methanol according to the suppliers instructions (Sigma, St Louis, MO). The methanol was evaporated by streaming nitrogen through the tube, and the powder was dissolved in 4 mg/ml fatty acid-free BSA in water to a final concentration of 125 µM. Wild-type Ax4 cells were allowed to develop on LPS saturated filters. At 5, 8 and 12 hours, filters were transferred to new pads saturated with either 5, 10 or 50 µM S-1-P in LPS, and development was allowed to proceed. The filters were photographed at 24 and 60 hours of development to characterize morphogenesis.
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RESULTS |
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Last, we tested two genes that are expressed at the terminal stage of development of prespore cells. The expression of the catB gene, which encodes the catalase B enzyme, which is expressed only in prespore cells (Garcia et al., 2000), and the spiA gene, which encodes a spore coat protein (Richardson and Loomis, 1992), is drastically reduced in the mutant. The diminished expression of these genes is consistent with the diminished ability of the mutant to complete morphogenesis and spore differentiation, though we cannot distinguish between diminished expression in all the cells versus normal expression in a small fraction of the cells.
Addition of extracellular S-1-P generates a phenocopy of the sglA null strain
The lack of S-1-P lyase suggested that the proximal cause of the developmental defects in the mutant were due to an increase in S-1-P. To examine this, we treated developing wild-type cells with extracellular S-1-P, added at different times during development (5, 8 and 12 hours), and followed the fate of the developing aggregates. Adding 5 or 10 µM S-1-P had no effect (data not shown). The addition of 50 µM S-1-P to wild-type cells led to abnormal development, which mimicked that of the S-1-P lyase null mutant (Fig. 11). The effect became more pronounced when the S-1-P was added at the later time points, and was not altered by prolonged incubation of up to 60 hours.
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DISCUSSION |
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The disruption of the S-1-P lyase probably results in elevated levels of S-1-P in the Dictyostelium cells. This is supported by the observation that platelets, which naturally lack S-1-P lyase activity, accumulate high levels of S-1-P (Yatomi et al., 1997), and by the observation that disruption of the yeast S-1-P lyase, BST1/DPL1, resulted in increased levels of S-1-P (Gottlieb et al., 1999; Saba et al., 1997). Thus, we have suggested that the disruption of the S-1-P lyase in our mutant results in an increase in S-1-P levels that counteracts the cytotoxic effects of cisplatin by promoting cell proliferation and inhibiting the induction of cell death (Li et al., 2000). Accordingly, we have now shown that deletion of the S-1-P lyase gene in Dictyostelium results in increased viability in stationary phase. In yeast, it has been shown that deletion of BST1/DPL1 results in the mutant cells growing to a higher cell density before entering stationary phase (Gottlieb et al., 1999). Thus, in both organisms, the deletion of this gene appears to confer a growth phase advantage.
Much less is known about the role of S-1-P in cell differentiation and multicellular development. Thus, it was significant that disruption of the Dictyostelium S-1-P lyase resulted in aberrant development. We have now analyzed this mutation throughout the entire developmental program. The data support the idea that its effects are highly pleiotropic, showing involvement throughout development, including the distribution of F-actin in aggregating cells, the ability to form migrating slugs, late developmental gene expression, and the ability to complete morphogenesis and spore differentiation. This array of phenotypes is consistent with the S-1-P lyase gene product functioning in a wide range of processes within the cell, and in accord with the expression of S-1-P lyase mRNA at a constant level throughout both mitotic growth and development.
The data indicate that the S-1-P lyase-null mutant is not entirely penetrant: some individual structures within the population do not display the mutant phenotype. Thus, a small percentage of aggregating cells have F-actin-filled pseudopods, some multicellular aggregates can make slugs with limited motility, and some spore specific gene expression and spore differentiation occurs. This behavior is consistent with the idea that the phenotype is the result of an increase in S-1-P, rather than the lack of a metabolite, as is often the case with gene disruptions. Heterogeneity in phenotypes that is due to incomplete genetic penetrance has been observed in numerous systems, including in a variety of human diseases and cancers.
Because S-1-P lyase is involved in metabolism of membrane components, it must be considered that these phenotypes could be the result of a global nonspecific alteration to the membrane structure. It is possible that the effects on F-actin distribution may be mediated at least partially through the membrane (see below). However, we feel that changes in membrane structure are not likely to be the cause for the other phenotypes in the mutant, owing to the known signal transduction roles of S-1-P and to the specificity and the independence of the phenotypes observed.
The finding that S-1-P lyase-null cells have altered shape and F-actin distribution is particularly exciting and relates directly to pharmacological studies with human cells. Exogenously added S-1-P has been shown to inhibit cell motility and chemotactic invasiveness of B16 melanoma (Sadahira et al., 1992) and estrogen-independent MDA-MB-231 invasive breast cancer cells (Sliva et al., 2000; Wang et al., 1999a). In addition, S-1-P inhibits motility of normal smooth muscle cells (Sadahira et al., 1992) and chemoattraction of neutrophils to interleukin 8 (Yamamura et al., 1996). Some data support the idea that S-1-P acts extracellularly through cell surface receptors to inhibit cell motility (Yamamura et al., 1997), although other studies support an intracellular mechanism for S-1-P. They show no involvement of EDG receptors, and that overexpression of the S-1-P kinase, which generates S-1-P, alters motility (Wang et al., 1999b). These changes in cell behavior are accompanied by a reduction in F-actin nucleation and pseudopod formation (Wang et al., 1999b; Yamamura et al., 1997). Dictyostelium discoideum has been the subject of intense investigation of cell motility and chemotaxis (Noegel et al., 1997; Parent and Devreotes, 1999), resulting in an unprecedented understanding of these responses in this organism. We are therefore well placed to use Dictyostelium to further elucidate the molecular roles of S-1-P in cell motility.
The inability to form migrating slugs is a dramatic phenotype that is associated with the disruption of the S-1-P lyase. It is possible that this defect is due to a fundamental defect in the motility of the individual cells, as reflected by the aberrant F-actin distribution. However, it is also possible that the end products of S-1-P degradation are needed for the synthesis of lipid components that are required for slugs to migrate. In this regard, it has been demonstrated that the modB-dependent form of protein glycosylation is required for slug migration (Alexander et al., 1988). Interestingly, some of the modB-modified proteins are also modified with a glycolipid anchor used for localization to the plasma membrane (Gooley et al., 1992). Indeed, phosphoethanolamine is a precursor of glycosylphosphatidylinositol, which functions as the membrane anchor (Canivenc-Gansel et al., 1998), and it is possible that the lipid anchors of these proteins are missing in the S-1-P lyase mutants, rendering the proteins nonfunctional.
The developmental timing of the S-1-P lyase null mutant is abnormal. The mutant aggregates slightly earlier than the wild type and is impaired in development. The expression of developmentally regulated genes reflects the phenotype, with a slightly premature expression of the CSA gene, and a lengthened period of expression of the ecmB and D19 genes. These results concur with many studies in Dictyostelium that have repeatedly demonstrated that the pattern of developmentally regulated gene expression is integrated with the overall progress of morphogenesis (Sussman and Brackenbury, 1976). That is, in slowly developing mutants, the periods of gene expression are lengthened, and in rapidly developing mutants, the periods are proportionally shortened.
The expression of both the catalase B gene and spiA spore coat protein gene is drastically reduced. Both these gene products are expressed at the very end of development in prespore cells. The absence of these gene products reflects that development in the mutant to the very late step of spore differentiation is impaired. The ability to form environmentally resistant spores is a major evolutionary advantage, and this result indicates the importance of the sglA gene in the natural history of this organism.
Extensive studies in Dictyostelium have shown that cAMP activation of PKA is required for normal cell differentiation and morphogenesis (Loomis, 1998). In animal cells, it has been shown that S-1-P can modulate the levels of cAMP (through modulation of adenylate cyclase activity) and that cAMP can, in turn, modulate the levels of S-1-P (through cAMP-mediated activation of sphingosine kinase) (Machwate et al., 1998; Van Brocklyn et al., 1998). This raises the possibility that some of the phenotypes that relate to spore specific gene expression and spore differentiation that we observe in the S-1-P lyase mutant may be mediated through the modulation of cAMP levels. Therefore, this work suggests another pathway for the regulation of developmental gene expression. However, changes in cAMP are probably not the underlying reason for other phenotypic changes in this mutant strain, including, for example, the F-actin distribution in single aggregation competent cells or the inhibition of slug migration.
Addition of exogenous S-1-P to wild-type cells mimics the overall aberrant developmental phenotype of the S-1-P lyase null mutant, and supports the idea that the mutant phenotypes result from increased levels of S-1-P in the cells. We do not know if the increased S-1-P level acts intracellularly or whether S-1-P is secreted and then acts in an autocrine manner. To date, the Dictyostelium genomic and cDNA sequencing projects have not identified an EDG receptor homolog in Dictyostelium. This may explain why relatively high levels of S-1-P are required to phenocopy the mutant, although Dictyostelium is well known for its relative resistance to a wide variety of drugs.
Overall, this study demonstrates that sphingolipids play a pivotal regulatory role in a wide range of processes in growth and multicellular development. This is reminiscent of the central regulatory role of cAMP during Dictyostelium development. Further work is required to elucidate all the details of how changes in sphingolipid metabolism affect development; the data presented here suggest that it has both structural and regulatory roles. To this end, we have recently identified other genes of this pathway, including two sphingosine kinases, two sphingosine-1-P phosphatases, an acid sphingomyelinase and a ceramidase, and we are currently constructing deletion and overexpression mutants with which to study the relationship between sphingolipids metabolism and multicellular development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Alexander, S., Smith, E., Davis, L., Gooley, A., Por, S. B., Browne, L. and Williams, K. L. (1988). Characterization of an antigenically related family of cell-type specific proteins implicated in slug migration in Dictyostelium discoideum. Differentiation 38, 82-90.[Medline]
Alexander, S., Sydow, L. M., Wessels, D. and Soll, D. R. (1992). Discoidin proteins of Dictyostelium are necessary for normal cytoskeletal organization and cellular morphology during aggregation. Differentiation 51, 149-161.[Medline]
Canivenc-Gansel, E., Imhof, I., Reggiori, F., Burda, P., Conzelmann, A. and Benachour, A. (1998). GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid. Glycobiology 8, 761-770.
Cocucci, S. and Sussman, M. (1970). RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45, 399-407.
Darcy, P. K., Wilczynska, Z. and Fisher, P. R. (1994). Genetic analysis of Dictyostelium slug phototaxis mutants. Genetics 137, 977-985.
Early, A. E., Williams, J. G., Meyer, H. E., Por, S. B., Smith, E., Williams, K. L. and Gooley, A. A. (1988). Structural characterization of Dictyostelium discoideum prespore specific gene D19 and of its product, cell surface glycoprotein PsA. Mol. Cell. Biol. 8, 3458-3466.[Medline]
Fisher, F. R., Smith, E. and Williams, K. L. (1981). An extracellular chemical signal controlling phototactic behavior by D. discoideum slugs. Cell 23, 799-807.[Medline]
Garcia, M. X. U., Foote, C., S., v. E., Devreotes, P. N., Alexander, S. and Alexander, H. (2000). Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light. Biochim. Biophys. Acta 1492, 295-310.[Medline]
Gooley, A. A., Marshchalek, R. and Williams, K. L. (1992). Size polymorphisms due to changes in the number of O-glycosylated tandem repeats in the Dictyostelium discoideum glycoprotein PsA. Genetics 130, 749-756.
Gottlieb, D., Heideman, W. and Saba, J. D. (1999). The DPL1 gene is involved in mediating the response to nutrient deprivation in Saccharomyces cerevisiae. Mol. Cell. Biol. Res. Commun. 1, 66-71.[Medline]
Igarashi, Y. (1997). Functional roles of sphingosine, sphingosine 1-phosphate, and methylsphingosines: in regard to membrane sphingolipid signaling pathways. J. Biochem. 122, 1080-1087.[Abstract]
Kamakura, T., Kobayashi, K., Tanaka, T., Yamaguchi, I. and Endo, T. (1987). Cloning and expression of a new structural gene for blasticidin S deaminase, a nucleoside aminohydrolase. Agric. Biol. Chem. 51, 3615-3618.
Kessin, R. H. (2001). Dictyostelium: Evolution, Cell Biology and the Development of Multicellularity. Cambridge, UK and New York: Cambridge University Press.
Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R. and Spiegel, S. (1998). Molecular cloning and functional characterization of murine sphingosine kinase. J. Biol. Chem. 273, 23722-23728.
Kuspa, A. and Loomis, W. F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc. Natl. Acad. Sci. USA 89, 8803-8807.[Abstract]
Laurence, A. and Firtel, R. (1999). Integration of signalling networks that regulate Dictyostelium differentiation. Annu. Rev. Cell Dev. Biol. 15, 469-517.[Medline]
Lee, S.-K., Yu, S.-L., Garcia, M. X., Alexander, H. and Alexander, S. (1997). Differential developmental expression of the repB and repD xeroderma pigmentosum related DNA helicase genes from Dictyostelium discoideum. Nucleic Acids Res. 25, 2365-2374.
Li, G., Alexander, H., Schneider, N. and Alexander, S. (2000). Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146, 2219-2227.
Loomis, W. F. (1998). Role of PKA in the timing of developmental events in Dictyostelium cells. Microbiol. Mol. Biol. Rev. 62, 684-694.
Machwate, M., Rodan, S. B., Rodan, G. A. and Harada, S. I. (1998). Sphingosine kinase mediates cyclic AMP suppression of apoptosis in rat periosteal cells. Mol. Pharmacol. 54, 70-77.
McRobbie, S. J., Jermyn, K. A., Duffy, K., Blight, K. and Williams, J. G. (1988). Two DIF inducible, prestalk specific messenger RNAs of Dictyostelium encode extracellular matrix proteins of the slug. Development 104, 275-284.[Abstract]
Morio, T., Urushihara, H., Saito, T., Ugawa, Y., Mizuno, H., Yoshida, M., Yoshino, R., Mitra, B., Pi, M., Sato, T., Takemoto, K., Yasukawa, R., Williams, J., Maeda, M., Takeuchi, I. et al. (1998). The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development. DNA Res. 5, 335-340.[Medline]
Muller, K. and Gerisch, G. (1978). A specific glycoprotein as the target site of adhesion blocking fab in aggregating Dictyostelium cells. Nature 274, 445-449.[Medline]
Newell, P. C., Telser, A. and Sussman, M. (1969). Alternative developmental pathways determined by environmental conditions in the cellular slime mold Dictyostelium discoideum. J. Bacteriol. 100, 763-768.[Medline]
Noegel, A. A., Rivero, F., Fucini, P., Bracco, E., Janssen, K. P. and Schleicher, M. (1997). Actin binding proteins: role and regulation. In Dictyostelium A Model System for Cell and Developmental Biology (ed. Y. Maeda, K. Inouye and I. Takeuchi), pp. 33-42. Tokyo: Universal Academy Press.
Okamoto, H., Takuwa, N., Gonda, K., Okazaki, H., Chang, K., Yatomi, Y., Shigematsu, H. and Takuwa, Y. (1998). EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition. J. Biol. Chem. 273, 27104-27110.
Parent, C. A. and Devreotes, P. N. (1999). A cells sense of direction. Science 284, 765-770.
Pyne, S. and Pyne, N. J. (1996). The differential regulation of cyclic AMP by sphingomyelin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in airway smooth muscle. Biochem. J. 315, 917-923.[Medline]
Pyne, S. and Pyne, N. J. (2000). Sphingosine 1-phosphate signalling in mammalian cells. Biochem. J. 349, 385-402.[Medline]
Rani, C. S., Wang, F., Fuior, E., Berger, A., Wu, J., Sturgill, T. W., Beitner-Johnson, D., LeRoith, D., Varticovski, L. and Spiegel, S. (1997). Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling. J. Biol. Chem. 272, 10777-10783.
Richardson, D. L. and Loomis, W. F. (1992). Disruption of the sporulation-specific gene spiA in Dictyostelium discoideum leads to spore instability. Genes Dev. 6, 1058-1070.[Abstract]
Saba, J. D., Nara, F., Bielawska, A., Garrett, S. and Hannun, Y. A. (1997). The BST1 gene of Saccharomyces cerevisiae is the sphingosine-1- phosphate lyase. J. Biol. Chem. 272, 26087-26090.
Sadahira, Y., Ruan, F., Hakomori, S. and Igarashi, Y. (1992). Sphingosine 1-phosphate, a specific endogenous signaling molecule controlling cell motility and tumor cell invasiveness. Proc. Natl. Acad. Sci. USA 89, 9686-9690.[Abstract]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sliva, D., Mason, R., Xiao, H. and English, D. (2000). Enhancement of the migration of metastatic human breast cancer cells by phosphatidic acid. Biochem. Biophys. Res. Commun. 268, 471-479.[Medline]
Soll, D. (1987). Methods for manipulating and investigating developmental timing in Dictyostelium discoideum. Methods Cell Biol. 28, 413-431.[Medline]
Spiegel, S. (1999). Sphingosine 1-phosphate: a prototype of a new class of second messengers. J. Leukoc. Biol. 65, 341-344.[Abstract]
Spiegel, S. and Milstien, S. (2000a). Functions of a new family of sphingosine-1-phosphate receptors. Biochim. Biophys. Acta 1484, 107-116.[Medline]
Spiegel, S. and Milstien, S. (2000b). Sphingosine-1-phosphate: signaling inside and out. FEBS Lett. 476, 55-57.[Medline]
Srinivasan, S., Griffiths, K. R., McGuire, V., Champion, A., Williams, K. L. and Alexander, S. (2000). The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum. Microbiology 146, 1829-1839.
Sussman, M. (1987). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol. 28, 9-29.[Medline]
Sussman, M. and Brackenbury, R. (1976). Biochemical and molecular-genetic aspects of cellular slime mold development. Annu. Rev. Plant Physiol. 27, 229-265.
Tolan, D., Conway, A. M., Pyne, N. J. and Pyne, S. (1997). Sphingosine prevents diacylglycerol signaling to mitogen-activated protein kinase in airway smooth muscle. Am. J. Physiol. 273, C928-C936.
Van Brocklyn, J. R., Lee, M. J., Menzeleev, R., Olivera, A., Edsall, L., Cuvillier, O., Thomas, D. M., Coopman, P. J., Thangada, S., Liu, C. H., Hla, T. and Spiegel, S. (1998). Dual actions of sphingosine-1-phosphate: extracellular through the Gi- coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J. Cell Biol. 142, 229-240.
van Veldhoven, P. P. and Mannaerts, G. P. (1993). Sphingosine-phosphate lyase. Adv. Lipid Res. 26, 69-98.[Medline]
Wang, F., Nohara, K., Olivera, A., Thompson, E. W. and Spiegel, S. (1999a). Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate. Exp. Cell Res. 247, 17-28.[Medline]
Wang, F., Van Brocklyn, J. R., Edsall, L., Nava, V. E. and Spiegel, S. (1999b). Sphingosine-1-phosphate inhibits motility of human breast cancer cells independently of cell surface receptors. Cancer Res. 59, 6185-6191.
Wessels, D. and Soll, D. R. (1998). Computer-assisted characterization of the behavioral defects of cytoskeletal mutants of Dictyostelium discoideum. In Motion Analysis of Living Cells (ed. D. R. Soll and D. Wessels), pp. 101-140. New York: Wiley-Liss.
Wu, J., Spiegel, S. and Sturgill, T. W. (1995). Sphingosine-1-phosphate rapidly activates the mitogen-activated protein kinase by a G protein-dependent mechanism. J. Biol. Chem. 270, 11484-11488.
Yamamura, S., Sadahira, Y., Ruan, F., Hakomori, S. and Igarashi, Y. (1996). Sphingosine-1-phosphate inhibits actin nucleation and pseudopodium formation to control cell motility of mouse melanoma cells. FEBS Lett. 382, 193-197.[Medline]
Yamamura, S., Yatomi, Y., Ruan, F., Sweeney, E. A., Hakomori, S. and Igarashi, Y. (1997). Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin- insensitive manner. Biochemistry 36, 10751-10759.[Medline]
Yatomi, Y., Yamamura, S., Ruan, F. and Igarashi, Y. (1997). Sphingosine 1-phosphate induces platelet activation through an extracellular action and shares a platelet surface receptor with lysophosphatidic acid. J. Biol. Chem. 272, 5291-5297.
Zada-Hames, I. M. and Ashworth, J. M. (1978). The cell cycle and its relationship to development in Dictyostelium discoideum. Dev. Biol. 63, 307-320.[Medline]