Aberrant stalk development and breakdown of tip dominance in Dictyosteliumcell lines with RNAi-silenced expression of calcineurin B
© Boeckeler et al; licensee BioMed Central Ltd. 2006
Received: 28 October 2005
Accepted: 02 March 2006
Published: 02 March 2006
Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, plays important roles in various cellular processes in lower and higher eukaryotes. Here we analyze the role of calcineurin in the development of Dictyostelium discoideum by RNAi-mediated manipulation of its expression.
The cnbA gene of Dictyostelium discoideum which encodes the regulatory B subunit (CNB) of calcineurin was silenced by RNAi. We found a variety of silencing levels of CNB in different recombinant cell lines. Reduction of CNB expression in a given cell line was correlated with developmental aberrations. Cell lines with strongly reduced protein levels developed slower than wild type cells and formed short stalks and spore heads with additional tips. Formation of short stalks results from incomplete vacuolization of prestalk cells during terminal differentiation. Expression of the stalk-specific gene ecmB was reduced in mutant cells. Aberrant stalk development is a cell autonomous defect, whereas the breakdown of tip dominance can be prevented by the presence of as low as 10% wild type cells in chimeras.
Silencing of calcineurin B in Dictyostelium by expression of RNAi reveals an unexpected link between increased intracellular calcium levels, possibly triggered by the morphogen DIF, activation of calcineurin, and the terminal stage of morphogenesis.
Calcineurin (CN), the Ca2+/calmodulin-dependent protein phosphatase, is highly conserved from yeast to mammalian cells. It consists of a catalytic (CNA) and a regulatory subunit (CNB) which form a heterodimer upon Ca2+ binding to CNB. The enzyme has been shown to play important roles in various cellular processes in lower and higher eukaryotes. In mammals these include T cell activation via dephosphorylation of the cytosolic component of nuclear factor of activated T cells (NFAT) , cardiac development and hypertrophy, learning and memory, and axonal pathfinding . In yeast calcineurin is involved in the regulation of ion homeostasis and cell cycle control . Whereas most organisms have at least two genes for the calcineurin subunits, the Dictyostelium discoideum genome contains single copy genes for CNA and CNB, whose expression changes during development of the organism. As in higher organisms CNB is required for high affinity binding of protein substrates by the holoenzyme . CNA is highest in vegetative cells and after aggregation [4, 5]. The coding mRNA for CNB is processed by an unorthodox mechanism starting during early development to give rise to a shorter isoform encoding a CNB protein with a truncated N terminus that does not contain the N myristoylation consensus site found in the full-length protein . Several attempts to delete the single gene for CNA (canA) in D. discoideum by insertion of selectable markers or to reduce its expression by antisense-mRNA failed (U. Kessen and R.M., unpublished results). 30-fold overexpression of the single cnbA gene which encodes the regulatory B subunit resulted in moderately accelerated multicellular development with recombinant populations completing morphogenesis about 2–3 hours earlier than wild type cells (F. Fouladi and B.W., unpublished observation). Pharmacological inhibition of D. discoideum CN using gossypol impaired growth and cellular signaling . Development of wild type cells treated with 25 μM gossypol was totally blocked for more than 24 hours. Studies using the classical inhibitors, Cyclosporin A (CsA) and FK506, indicated that CN regulates Ca2+ stress-induced transcription activation of the Ca2+-ATPase patA gene  as well as expression of the "calcium upregulated" (Cup) class of Ca2+-binding proteins . In an in vitro differentiation assay CsA and FK506 strongly inhibited stalk cell formation in the wild-type and spore formation in a sporogenous Dictyostelium strain . These agents also reduced the expression of prestalk and prespore-specific transcripts, assigning a function for CN as a general activator of differentiation
During development D. discoideum cells aggregate in response to cAMP, form slugs and differentiate into two major cell types, prespore and prestalk cells. These cells organize in a spatial pattern with the prespore region localizing to the posterior and the organizer, prestalk region to the anterior of the slug. Development is completed by the formation of fruiting bodies consisting of a stalk of dead cells and the spore head. Several lines of evidence link the level of cytosolic Ca2+ (Ca2+i) to cell type differentiation. Cells in S and early G2 cell cycle phases, which show a tendency to become prestalk cells, have high intracellular calcium . At the slug stage, a gradient of Ca2+i along the anterior-posterior axis has been found which is inversely correlated with the amount of sequestered Ca2+ and the calcium-sequestering activity in high-capacity Ca2+ stores . High Ca2+i has been shown to be necessary for induction of the prestalk-specific gene ecmB by the morphogen, differentiation-inducing factor (DIF), and a sustained increase in Ca2+i was proposed to be responsible for prestalk-specific gene expression . This work also showed that the increase in Ca2+i is brought about by DIF. It is unclear how DIF increases Ca2+i, and the mechanism by which Ca2+i ultimately leads to activation of ecmB expression has remained elusive.
RNA interference-mediated gene silencing was recently established in Dictyostelium . RNAi-mediated silencing of gene expression is induced by double-stranded RNA and its processing to 23 mers which cause the degradation of endogenous target mRNAs [14, 15]. The technique appears to be well suited for the analysis of potentially essential genes in the haploid Dictyostelium genome since it can lead to different levels of silencing from almost wild type to maximally reduced levels in individual cell lines. This would allow to determine the repression level of a given gene that is just tolerable for cell survival.
We show here that a hairpin RNA construct targeted against cnbA effectively reduces the cellular CNB concentration. Recombinant cell lines showed retardation of multicellular development, culminating in fruiting bodies with short stalks and the formation of additional tips on the spore mass of developing fruiting bodies.
Isolation of cnbARNAi transgenic cell lines
Downregulation of calcineurin B
Characterization of cnbA-RNAi mutant 01–5
Cell lines with slight reduction of CNB expression displayed no or only mild phenotypic aberrations which were mainly manifest in the erection of fruiting bodies with somewhat shorter stalks than observed with wild type organisms (not shown). However, phenotypic alterations were much more prominent in cell lines with pronounced silencing of CNB. To characterize the phenotypic consequences of silencing of cnbA mRNA 10 independent transformants with drastically reduced expression of CNB were further analyzed. The phenotypes of all these transformants were very similar to cell line 01–5 which is described in more detail below.
We first compared expression of cnbA mRNA in cell line 01–5 and parental AX2 cells by Northern blotting. Figure 4 shows that the recombinant cell line had drastically reduced amounts of endogenous cnbA mRNA (0.7 kb). A strong signal corresponding to the size of the RNAi construct (1.2 kb) indicates massive expression of the transfected gene. Degradation of both RNAi and endogenous cnbA mRNA is indicated by the smear detected in the mRNA isolated from 01–5 cells.
Cell-autonomous and non cell-autonomous effects of RNAi expression
Analysis of aberrant stalks by light and electron microscopy
The defect in stalk formation which was observed in cell lines with strongly reduced CNB expression occurs due to smaller and incompletely vacuolated stalk cells and this result points to an important role for CNB or the CN holoenzyme in the regulation of stalk development in Dictyostelium. DIF-1 induces the expression of prestalk-specific genes via an increase in the intracellular free Ca2+ . Based on our results we speculate that the effects of increased Ca2+i are mediated by CN. In analogy to the well-characterized role of CN in dephosphorylation of transcription factor complexes of the NFAT type we propose that calcineurin, in response to an increase in cytosolic Ca2+, dephosphorylates a cytosolic component of a transcription complex, which is then translocated to the nucleus and activates – in concert with nuclear components of the active complex – the transcription of prestalk genes. This hypothesis is directly supported by our observation that RNAi mutant 01–5 failed to properly induce expression of the prestalk-specific gene ecmB during development.
The phenotypic defects which we report here complement previous studies on the effects of mutations in the Dictyostelium glycogen synthase kinase-3 homolog GskA  which has been shown to promote posterior cell patterning and inhibit anterior cell differentiation [reviewed in ]. GskA is regulated through an intracellular protein tyrosine kinase/phosphatase pathway mediated by the cAMP receptors CAR1 and/or CAR3 and CAR4. GskA is activated by CAR1- and/or CAR3-mediated activation of the tyrosine kinase ZAK1 , and inactivated by CAR4-mediated activation of a protein tyrosine phosphatase . GskA null cells show accelerated early development and form only stalk cells during morphogenesis . Both the gskA null and the cnbA RNAi phenotypes are cell autonomous ; see above, fig. 8A. The direct link between GskA and CN is supported by recent results that come from analysis of the NFAT pathway. In T cells phosphorylation of NFAT by GSK3 represses NFAT-dependent gene expression by inhibition of NFAT binding to DNA . Inhibition of re-export of NFAT to the cytosol when GSK3 is inactivated, leads to increased NFAT-dependent activation of gene expression .
Deletion of the Dictyostelium aarA gene, a homolog of mammalian β-catenin, leads to a closely similar breakdown of tip dominance and to formation of ectopic tips during late development as silencing of CNB. In both cases, the defect is non-cell autonomous ; see above, fig. 8B. It has been shown that AarA plays a necessary role in the formation of adherence junctions at the neck of the rising stalk . β-catenin is also a transcriptional co-activator in the metazoan canonical Wnt pathway. In the control of dorsoventral axis formation in Xenopus by extracellular signals, a CN-independent NFAT mutant inhibited anterior development of the primary axis, whereas a dominant negative NFAT mutation induced ectopic dorsal axis formation and the expression of target genes of the canonical Wnt pathway, suggesting that CN and NFAT are part of the noncanonical Wnt/Ca2+ pathway which leads to inhibition of the canonical Wnt pathway upstream of β-catenin  (for a Wnt/Ca2+ pathway overview see ). It is therefore possible to propose a model for Dictyostelium tip dominance where a pathway similar to the canonical metazoan Wnt pathway is acting through CAR1/CAR3 to activate GskA, and a noncanonical "Wnt/Ca2+" pathway through CAR4 to activate CN via an intracellular Ca2+ signal. The postulated transcription factor target for CN in Dictyostelium is so far unknown.
Calcineurin B is a homolog of members of the 4 EF hand calcium sensor protein family which include, for example, recoverin, the neuronal calcium sensor-1 (NCS-1), and the plant calcineurin B-like proteins [(CBLs ]. It has recently been demonstrated that N myristoylation, a common feature of CNB and the Ca2+ sensor protein family, increases the cooperativity of Ca2+ binding to NCS-1 and leads to larger conformational changes upon Ca2+ binding than in the nonmyristoylated protein . If Ca2+ sensing is indeed a major function of calcineurin B, the phenotypic defects observed in our cnbA RNAi mutants may be due to the failure to correctly sense the slow, sustained increase in Ca2+i from ca. 50 to ca. 150 nM during a period of about 8 hours , which was proposed to induce prestalk gene expression. Starting during early development D. discoideum cells express two CNB isoforms, one of them lacking the myristoylation consensus site. Further work has to show whether it is the myristoylated CNB isoform which plays a crucial role in the mediation of DIF effects on prestalk gene expression.
Dictyostelium calcineurin B knock down mutants produced smaller stalk cells and as a consequence smaller stalks due to incomplete vacuolization of stalk cells. We propose that calcineurin mediates the effects of a differentiation-inducing factor (DIF)-elicited increase in free intracellular calcium in prestalk cells via the dephosphorylation of a cytosolic transcription complex component which activates the expression of prestalk genes. Culminating fruiting bodies formed ectopic tips on the spore heads which points to a link between calcineurin and the wnt pathway in the social amoebae.
Growth and development of Dictyostelium discoideum
D. discoideum AX2 cells were grown at 22°C in AX medium . Development was induced by washing cells twice with ice-cold Sørensen phosphate (SP) buffer [17 mM (KH2/Na2H)PO4], pH 6.0 and spreading 2 × 107 cells on HABP nitrocellulose filters (0.45 μm, diameter 47 mm, Millipore, Eschborn, Germany) supported by two paper filters (diameter 47 mm) soaked with buffer.
Transformation of Dictyostelium discoideum
D. discoideum AX2 cells were grown in AX medium to a density of 5 × 106 cells/ml, washed twice with ice-cold H-50 buffer, resuspended at 2 × 107 cells/ml, and 100 μl of this suspension was electroporated with 10 μg of plasmid DNA [31, 32]. Transformed cells were grown on suitable selective media and clonal populations were obtained by serial dilution in microtiter plates. In initial experiments we observed that the phenotypic aberrations which correlated with RNAi expression became gradually weaker upon serial passages of the cells. Therefore, immediately after isolation, clones of recombinant cells were grown to final numbers of approx. 1.5 × 109 (ca. 31 to 32 generations) and allowed to form spores on SP agar plates which were harvested and kept in SP buffer at -70°C until used to inoculate experimental cultures. These underwent additional 13 to 15 generations.
Construction of plasmids
cnbA cDNA was amplified by reverse transcription of total RNA from vegetative D. discoideum using the primer pair 5'GGGCCATGGGGAATCAACATTCATTATTA3' and 5'TTATTCTGACCAATTTACGCTAAG3'. The product was cloned into pDrive (Qiagen, Hilden, Germany) to obtain plasmid pBW104. Construction of the RNAi-encoding plasmid for cnbA was done in three steps. First, a fragment of 437 bp amplified with primers 5'CCGGATCCAAGCTTAAAGAACAATTAGAACAAATGA3' and 5'CCGTCGACTCACCTTCAATAATAGTTTTATCAACA3' was cloned in sense orientation into BamHI/SalI-digested pUC18 , yielding pKB05. Second, pKB06 was generated by ligating a longer cnbA fragment of 538 bp, amplified with primers 5'CCCTGCAGAAAGAACAATTAGAACAAATGA3' and 5'CCGTCGACTTCTGACCAATTTACGCTAAGTTTT3', in reverse orientation into pKB05 after digestion with PstI and SalI. Finally the fused fragments were subcloned into PstI/BamHI-digested pDNeoII  to obtain pKB07 (see below, fig. 1).
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), chromatographed on 0.9% agarose gels containing 6.6% formaldehyde and blotted onto nylon membranes which were hybridized with DIG-labelled cDNA probes and stained with CSPstar as recommended by the manufacturer (all reagents from Roche Molecular Diagnostics, Mannheim, Germany).
SDS/polyacrylamide gel electrophoresis and Western blotting
Proteins were chromatographed on 15% polyacrylamide/0.1% SDS gels , transferred to nitrocellulose membranes and probed with 1:5000 diluted rabbit antiserum against recombinant D. discoideum calcineurin B as described .
Analysis of developmental chimeras
AX2 wild type and mutant strain 01–5 were cultured in AX medium. Cells were washed twice in SP buffer and resuspended at 2 × 107 cells/ml. The cells were mixed in different ratios and 2 × 107 cells spread on HABP nitrocellulose filters. Filters were incubated in the dark at 22°C for 30 h.
Analysis of fruiting body size and spore viability
Wild type and mutant strains were cultured in AX medium. Cells were washed twice in SP buffer and resuspended to 4 × 106 cells/ml. 100 μl each were spread into individual wells of a 96 well microtiter plate filled with 200 μl SP agar each. Organisms were allowed to develop for 48 h, fruiting bodies were collected, resuspended in SP buffer and frozen at -70°C. After one day spores were spread on nutrient agar with Klebsiella planticola, incubated for three days at 22°C and the numbers of clones were counted.
Embedding of fruiting bodies
Dictyostelium fruiting bodies developed on HABP filters were fixed for 3 days in a humid chamber with glutaraldehyde (2.5%) in 0.1 M Na/K phosphate buffer (pH 7.0), using both fixative solution (completely absorbed by the HABP filter) and the arising fixative vapour. The fruiting bodies were then embedded in 1.5% low melting agarose (LGT Agarose, Marine Colloids Inc., US) at 20°C. After hardening the agarose by cooling down to 0°C, blocks including fruiting bodies were prepared. The specimens were then fixed a second time for 2 h at RT with glutaraldehyde in 0.1 M Na/K phosphate buffer (pH 7.0) containing paraformaldehyde (2%) and tannic acid (0.2%). After washing 3 times with the same buffer for 20 min each a third fixation with osmium tetroxyde (1% in 50 mM Na/K phosphate buffer, pH 7.0) for 12 h at 20°C was performed, followed by repeated washing with the same buffer. The specimens were dehydrated in a graded series of ethanol. They were incubated in propylene oxide, followed by a mixture of propylene oxide/epoxide resin (v/v) and pure epoxide resin ERL . The resin was polymerized for 2 days at 60°C.
Transmission electron microscopy
Ultrathin sections of ca. 50–80 nm were prepared and mounted on 400 mesh cupper grids. The samples were contrasted with 2% w/v uranyl acetate for 10 min and with 0.2% w/v lead citrate in 0.2 M NaOH for 2 min. Electron micrographs were obtained with a Zeiss EM 10 microscope using Scientia negative films. The negatives were scanned with an Epson 1680 Pro scanner at a resolution of 1200 dpi.
List of abbreviations used
neuronal calcium sensor-1
nuclear factor of activated T cells
glycogen synthase kinase.
We are grateful to Angela Lahrz for skilled technical assistance and to Mirko Trilling for help in the construction of pBW104. We thank Freya Kaulbars for help in microscopical analyses, Petra Dersch for critically reading the manuscript and Wolfgang Nellen for most helpful suggestions and for communicating unpublished results. This work was supported by the Deutsche Forschungsgemeinschaft (grant MU 1286/3).
- Hogan PG, Chen L, Nardone J, Rao A: Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003, 17: 2205-2232. 10.1101/gad.1102703.View ArticlePubMedGoogle Scholar
- Crabtree GR: Calcium, calcineurin, and the control of transcription. J Biol Chem. 2001, 276: 2313-2316. 10.1074/jbc.R000024200.View ArticlePubMedGoogle Scholar
- Rusnak F, Mertz P: Calcineurin: form and function. Physiol Rev. 2000, 80: 1483-1521.PubMedGoogle Scholar
- Aichem A, Mutzel R: Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium. J Mol Biol. 2001, 308: 873-882. 10.1006/jmbi.2001.4645.View ArticlePubMedGoogle Scholar
- Dammann H, Hellstern S, Husain Q, Mutzel R: Primary structure, expression and developmental regulation of a Dictyostelium calcineurin A homologue. Eur J Biochem. 1996, 238: 391-399. 10.1111/j.1432-1033.1996.0391z.x.View ArticlePubMedGoogle Scholar
- Weissenmayer B, Boeckeler K, Lahrz A, Mutzel R: The calcineurin inhibitor gossypol impairs growth, cell signalling and development in Dictyostelium discoideum. FEMS Microbiol Lett. 2005, 242: 19-25. 10.1016/j.femsle.2004.10.035.View ArticlePubMedGoogle Scholar
- Moniakis J, Coukell MB, Janiec A: Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum. J Cell Sci. 1999, 112: 405-414.PubMedGoogle Scholar
- Coukell B, Li Y, Moniakis J, Cameron A: The Ca2+/calcineurin-regulated cup gene family in Dictyostelium discoideum and its possible involvement in development. Eukaryot Cell. 2004, 3: 61-71. 10.1128/EC.3.1.61-71.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Horn F, Gross J: A role for calcineurin in Dictyostelium discoideum development. Differentiation. 1996, 60: 269-275. 10.1046/j.1432-0436.1996.6050269.x.View ArticlePubMedGoogle Scholar
- Azhar M, Kennady PK, Pande G, Espiritu M, Holloman W, Brazill D, Gomer RH, Nanjundiah V: Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum. Int J Dev Biol. 2001, 45: 405-414.PubMedGoogle Scholar
- Schlatterer C, Walther P, Muller M, Mendgen K, Zierold K, Knoll G: Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells. Cell Calcium. 2001, 29: 171-182. 10.1054/ceca.2000.0181.View ArticlePubMedGoogle Scholar
- Schaap P, Nebl T, Fisher PR: A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium. EMBO J. 1996, 15: 5177-5183.PubMed CentralPubMedGoogle Scholar
- Martens H, Novotny J, Oberstrass J, Steck TL, Postlethwait P, Nellen W: RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase. Mol Biol Cell. 2002, 13: 445-453. 10.1091/mbc.01-04-0211.PubMed CentralView ArticlePubMedGoogle Scholar
- Hannon GJ: RNA interference. Nature. 2002, 418: 244-251. 10.1038/418244a.View ArticlePubMedGoogle Scholar
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998, 391: 806-811. 10.1038/35888.View ArticlePubMedGoogle Scholar
- Eichinger L, Pachebat JA, Glockner G, Rajandream MA, Sucgang R, Berriman M, Song J, Olsen R, Szafranski K, Xu Q, Tunggal B, Kummerfeld S, Madera M, Konfortov BA, Rivero F, Bankier AT, Lehmann R, Hamlin N, Davies R, Gaudet P, Fey P, Pilcher K, Chen G, Saunders D, Sodergren E, Davis P, Kerhornou A, Nie X, Hall N, Anjard C, Hemphill L, Bason N, Farbrother P, Desany B, Just E, Morio T, Rost R, Churcher C, Cooper J, Haydock S, vanDriessche N, Cronin A, Goodhead I, Muzny D, Mourier T, Pain A, Lu M, Harper D, Lindsay R, al : The genome of the social amoeba Dictyostelium discoideum. Nature. 2005, 435: 43-57. 10.1038/nature03481.PubMed CentralView ArticlePubMedGoogle Scholar
- Kessin RH: Dictyostelium - Evolution, cell biology, and the development of multicellularity. 2001, Cambridge, UK, Cambridge Univ. Press, xiv + 294-View ArticleGoogle Scholar
- Harwood AJ, Plyte SE, Woodgett J, Strutt H, Kay RR: Glycogen synthase kinase 3 regulates cell fate in Dictyostelium. Cell. 1995, 80: 139-148. 10.1016/0092-8674(95)90458-1.View ArticlePubMedGoogle Scholar
- Chisholm RL, Firtel RA: Insights into morphogenesis from a simple developmental system. Nat Rev Mol Cell Biol. 2004, 5: 531-541. 10.1038/nrm1427.View ArticlePubMedGoogle Scholar
- Ginsburg GT, Kimmel AR: Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium. Genes Devel. 1997, 11: 2112-2123.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim L, Harwood A, Kimmel AR: Receptor-dependent and tyrosine phosphatase-mediated inhibition of GSK3 regulates cell fate choice. Dev Cell. 2002, 3: 523-532. 10.1016/S1534-5807(02)00269-1.View ArticlePubMedGoogle Scholar
- Neal JW, Clipstone NA: Glycogen Synthase Kinase-3 Inhibits the DNA Binding Activity of NFATc. J Biol Chem. 2001, 276: 3666-3673. 10.1074/jbc.M004888200.View ArticlePubMedGoogle Scholar
- Diehn M, Alizadeh AA, Rando OJ, Liu CL, Stankunas K, Botstein D, Crabtree GR, Brown PO: Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation. Proc Natl Acad Sci U S A. 2002, 99: 11796-11801. 10.1073/pnas.092284399.PubMed CentralView ArticlePubMedGoogle Scholar
- Coates JC, Grimson MJ, Williams RS, Bergman W, Blanton RL, Harwood AJ: Loss of the beta-catenin homologue aardvark causes ectopic stalk formation in Dictyostelium. Mech Dev. 2002, 116: 117-127. 10.1016/S0925-4773(02)00152-1.View ArticlePubMedGoogle Scholar
- Grimson MJ, Coates JC, Reynolds JP, Shipman M, Blanton RL, Harwood AJ: Adherens junctions and beta-catenin-mediated cell signalling in a non-metazoan organism. Nature. 2000, 408: 727-731. 10.1038/35047099.View ArticlePubMedGoogle Scholar
- Saneyoshi T, Kume S, Amasaki Y, Mikoshiba K: The Wnt/calcium pathway activates NF-AT and promotes ventral cell fate in Xenopus embryos. Nature. 2002, 417: 295-299. 10.1038/417295a.View ArticlePubMedGoogle Scholar
- Huelsken J, Behrens J: The Wnt signalling pathway. J Cell Sci. 2002, 115: 3977-3978. 10.1242/jcs.00089.View ArticlePubMedGoogle Scholar
- Albrecht V, Weinl S, Blazevic D, D'Angelo C, Batistic O, Kolukisaoglu U, Bock R, Schulz B, Harter K, Kudla J: The calcium sensor CBL1 integrates plant responses to abiotic stresses. Plant J. 2003, 36: 457-470. 10.1046/j.1365-313X.2003.01892.x.View ArticlePubMedGoogle Scholar
- Jeromin A, Muralidhar D, Parameswaran MN, Roder J, Fairwell T, Scarlata S, Dowal L, Mustafi SM, Chary KV, Sharma Y: N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1. J Biol Chem. 2004, 279: 27158-27167. 10.1074/jbc.M312172200.View ArticlePubMedGoogle Scholar
- Watts DJ, Ashworth JM: Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J. 1970, 119: 171-174.PubMed CentralView ArticlePubMedGoogle Scholar
- Schlatterer C, Knoll G, Malchow D: Intracellular calcium during chemotaxis of Dictyostelium discoideum - a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements. Eur J Cell Biol. 1992, 58: 172-181.PubMedGoogle Scholar
- Pang KM, Lynes MA, Knecht DA: Variables controlling the expression level of exogenous genes in Dictyostelium. Plasmid. 1999, 41: 187-197. 10.1006/plas.1999.1391.View ArticlePubMedGoogle Scholar
- Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985, 33: 103-119. 10.1016/0378-1119(85)90120-9.View ArticlePubMedGoogle Scholar
- Witke W, Nellen W, Noegel A: Homologous recombination in the Dictyostelium alpha-actinin gene leads to an altered mRNA and lack of the protein. EMBO J. 1987, 6: 4143-4148.PubMed CentralPubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticlePubMedGoogle Scholar
- Spurr AR: A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969, 26: 31-43. 10.1016/S0022-5320(69)90033-1.View ArticlePubMedGoogle Scholar
- Richardson KC, Jarett L, Finke EH: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 1960, 35: 313-323.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.