UBC Faculty Research and Publications

A novel Dictyostelium RasGEF required for chemotaxis and development Arigoni, Maddalena; Bracco, Enrico; Lusche, Daniel F; Kae, Helmut; Weeks, Gerald; Bozzaro, Salvatore Dec 7, 2005

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-12860_2005_Article_169.pdf [ 3.1MB ]
JSON: 52383-1.0132663.json
JSON-LD: 52383-1.0132663-ld.json
RDF/XML (Pretty): 52383-1.0132663-rdf.xml
RDF/JSON: 52383-1.0132663-rdf.json
Turtle: 52383-1.0132663-turtle.txt
N-Triples: 52383-1.0132663-rdf-ntriples.txt
Original Record: 52383-1.0132663-source.json
Full Text

Full Text

ralssBioMed CentBMC Cell BiologyOpen AcceResearch articleA novel Dictyostelium RasGEF required for chemotaxis and developmentMaddalena Arigoni1, Enrico Bracco1, Daniel F Lusche2, Helmut Kae3, Gerald Weeks3 and Salvatore Bozzaro*1Address: 1Department of Clinical and Biological Sciences, University of Torino, Regione Gonzole 10, 10043 Orbassano, Italy, 2Faculty of Biology, University of Konstanz, 78457 Konstanz, Germany and 3Dept. Microbiology and Immunology, University of British Columbia, Canada V6T1Z3Email: Maddalena Arigoni - maddalena.arigoni@unito.it; Enrico Bracco - enrico.bracco@unito.it; Daniel F Lusche - Daniel.Lusche@web.de; Helmut Kae - hkae@interchange.ubc.ca; Gerald Weeks - gweeks@interchange.ubc.ca; Salvatore Bozzaro* - salvatore.bozzaro@unito.it* Corresponding author    AbstractBackground: Ras proteins are guanine-nucleotide-binding enzymes that couple cell surfacereceptors to intracellular signaling pathways controlling cell proliferation and differentiation, bothin lower and higher eukaryotes. They act as molecular switches by cycling between active GTP andinactive GDP-bound states, through the action of two classes of regulatory proteins: a) guaninenucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wideanalysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of RasGuanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteinsby catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activatorof Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth ordevelopment, have been characterized in Dictyostelium. We report here the identification andcharacterization of a fifth non-redundant GEF, RasGEFM.Results: RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP,RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growthand development. Inactivation of the gene results in cells that form small, flat aggregates and fail todevelop further. Expression of genes required for aggregation is delayed. Chemotaxis towardscAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMPaccumulates during early development to a much lower extent than in wild type cells. Adenylylcyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase isstimulated to higher levels than in the wild type. The actin polymerization response to cAMP is alsoaltered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitroexperiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the Gprotein.Conclusion: The data indicate that RasGEFM is involved in the establishment of the cAMP relaysystem. We propose that RasGEFM is a component of a Ras regulated pathway, which integratesignals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase.Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results inPublished: 07 December 2005BMC Cell Biology 2005, 6:43 doi:10.1186/1471-2121-6-43Received: 16 June 2005Accepted: 07 December 2005This article is available from: http://www.biomedcentral.com/1471-2121/6/43© 2005 Arigoni et al; licensee 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.Page 1 of 18(page number not for citation purposes)altered chemotaxis.BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43BackgroundThe ras proto-oncogenes encode membrane-bound smallmonomeric GTP-binding proteins with molecular massesranging between 20 to 40 kDa, which are highly con-served in the course of eukaryotic evolution. Ras proteinscontrol fundamental cell processes including prolifera-tion, differentiation, motility and polarity [1-3]. Like het-erotrimeric G proteins they act as molecular switchescycling between two interconvertible forms: inactive,when bound to guanosine diphosphate (GDP), andactive, when bound to guanosine triphosphate (GTP) [4].Conversion between GTP and GDP-bound states is tightlyregulated by two set of proteins: guanosine-nucleotideexchange factors (GEFs), and GTPase activating proteins(GAPs). GEF proteins cause activation by catalysing theexchange of bound GDP with GTP, whereas GAPs inacti-vate Ras by increasing their rate of GTP hydrolysis [5].Systems composed by GTPases, GAPs and GEFs allowgreat versatility in the construction of signaling pathways.Signals can be amplified (one GEF could activate severalGTPases), integrated (several pathways activate the sameGEF and GAP, and the behaviour of one GTPase dependson the total effect of all its GEFs and GAPs), or split (oneGTPase induces many effects) [6]. This versatility allowssmall GTPases to mediate a wide range of different biolog-ical functions among different organisms. For example inS. cerevisiae RasGEF CDC25 is required for Ras mediatedactivation of adenylyl cyclase and it is essential for prolif-eration and spore germination [7], whereas in Drosophila,the RasGEF Son-of-sevenless (Sos) functions upstream ofRas in R7 photoreceptor differentiation [8].Despite its relatively small genome, Dictyostelium pos-sesses a relatively large number of ras and rasGEF encod-ing genes. Dictyostelium is a lower eukaryote with a simplelife cycle, consisting of growth and multicellular develop-ment, the latter being fully completed in approximately24 hours. The amoebae live as single cells, growing byfeeding on bacteria, which are taken up by phagocytosis,and dividing by binary fission. Upon starvation, cells startreleasing the chemoattractant cAMP and gather by chem-otaxis to form multicellular aggregates. Within each aggre-gate, cells differentiate into prespore and several classes ofprestalk cells, while undergoing a series of morphogeneticchanges, which end up in the formation of fruiting bodies.Fruiting bodies consist of slender stalks of vacuolated,dead cells, bearing on top spores encapsulated in sori.Here we report the identification and characterization of anovel RasGEF, named RasGEFM. To date only four Dicty-ostelium RasGEFs have been characterized, namely ras-GEFA, formerly known as aimless [9], rasGEFB [10], gbpCcyclic nucleotide-binding domain, which is associatedwith a MAPKKK-like kinase domain, Leucine Rich Repeats(LRR) and a Ras domain. GbpD is highly similar to GbpC,but it lacks the Ras, MAPKKK-like and the LRR domains[11]. These two proteins control myosin phosphorylationand, as consequence, cell motility and chemotaxis [12].The GEF protein encoded by the rasGEFA gene is essentialfor cell aggregation, acting at the level of adenylyl cyclaseactivation [9]. Dictyostelium cells lacking rasGEFB aredefective in early development, although they eventuallyform tiny but normally proportioned fruiting bodies. Fur-thermore, these cells move unusually rapidly and showsevere impairment in cell growth [10].Here we present evidences that RasGEFM is involved in aRas regulatory network, required for cAMP receptordependent signal transduction. Mutant cells lacking therasGEFM gene produce very small, flat aggregates and failto develop further. Chemotaxis is altered in the mutant,due to inability of the cells to polarize properly. The phe-notype can be partially rescued by pulsing cells withcAMP. We show that RasGEFM is involved in controllingcAMP relay and cell motility.ResultsIdentification, cloning and sequence analysis of D.d.rasGEFMTo identify Ras regulator proteins in the Dictyosteliumgenome a bioinformatic approach was taken, based on atBLASTn algorithm, to search the Dictyostelium genomeproject databases [13] and [14]. Using known RasGEFsdomains as query, we identified approximately 30 genesencoding for proteins with significant homology to puta-tive RasGEF proteins (unpublished results). Among theputative RasGEF encoding genes, one of these, designatedas Dd rasGEFM, was isolated for functional studies. TherasGEFM gene, located on the chromosome 2, is organ-ized in 4 exons, which are interrupted by 3 introns. South-ern blot analyses performed under high and lowstringency conditions, and extensive analysis of the Dicty-ostelium genomic sequence databases, indicated that thegene is present as single locus (data not shown). The ras-GEFM gene encodes a relatively large multi-domain pro-tein of 929 amino acids, with a calculated molecular massof approximately 102 kDa, whose domain structure is dis-played in Fig. 1. The amino-terminal region of the proteincontains six short poly-proline stretches, which are puta-tive consensus docking site for SH3 or WW domains [15]whereas the carboxy-terminal region possesses the puta-tive RasGEF catalytic domain. BLAST analysis shows thatthe catalytic domain, with evolutionary identical aminoacid residues conserved, shares high homology with themurine p140GRF (30% identity, 59% homology). AmongPage 2 of 18(page number not for citation purposes)and gbpD [11]. The latter two encode for unconventionalRasGEFs. GbpC possesses a RasGEF domain coupled to aDictyostelium RasGEF proteins, the most similar to Ras-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43GEFM are two putative uncharacterized RasGEFs (Ras-GEFE and RasGEFJ) (Fig. 1).Between the catalytic domain and the amino-terminalpoly-proline rich region, there are a DEP and a RasGEF-Nterminal domain. The RasGEF-N terminal module ispeculiar only for Ras specific GEFs, and it is likely to havea purely structural role [16]. The DEP (Dishevelled-Eglin-Pleckstrin homology) domain, located between the cata-lytic and the RasGEF-N terminal domain, shows signifi-cant homology to the DEP domain of the humanpleckstrin 25% overall homology and 11% identity, ver-sus a 27% overall homology and 14% identity displayedby the Dictyostelium GbpC DEP [11] when compared tothe human plekstrin counterpart. DEP is a widespreadmotif found in proteins involved in Wnt signalling, in reg-ulators of G protein signalling (RGS), in pleckstrin andother signalling proteins [17,18], responsible either fortargeting proteins to the membrane or mediating protein-rasGEFM gene expression is developmentally regulated and partially controlled by the G proteinOne of the prominent features of Dictyostelium life cycle isthe transition from solitary amoebae to multicellularaggregates. This transition is triggered by starvation of thecells, is enhanced by periodic release of cAMP, and resultsin the coordinated activation and repression of aggrega-tion-specific and growth-phase genes, respectively [19].We examined the expression pattern of the rasGEFM geneduring development of wild-type cells and in threemutant strains, which are blocked at sequential steps ofdevelopment. Two transcripts were detected in Northernblots: the upper one is present during growth and at thebeginning of development and disappears at 6 hours ofstarvation. The lower transcript is barely visible at thebeginning of starvation, reaches its maximal expression at6 hours of development and decreases thereafter, thoughbeing present up to the end of development (Fig. 2A).Schematic representation of the different functional domains of RasGEFM identified with MotifScanFigure 1Schematic representation of the different functional domains of RasGEFM identified with MotifScan. The Ras-GEFM protein contains the following recognizable modules: 6 proline rich regions, a RasGEF N-terminal domain, a DEP domain and a RasGEF catalytic domain. The sequence of the RasGEFM catalytic domain (aminoacids 673–863) has been aligned, using the ClustaIW program, with representative RasGEFs proteins from different organisms and others putative Dictyostelium Ras-GEFs. Identical and conserved aminoacid residues are boxed in dark or light gray respectively. Accession numbers are referred to GeneBank: [AAN46882 D.d.GEF M, AAN46874 D.d.GEF E, AAN46879 D.d.GEF J, A41216 D.m.Sos, P28818 R.n.p140, A38985 H.s. CDC25]Page 3 of 18(page number not for citation purposes)protein interaction, although the underlying molecularmechanism remains still unknown.We examined rasGEFM expression in the LW6 mutantlacking the β subunit of heterotrimeric G protein [20].BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43Although two Gβ genes are present in the Dictyosteliumgenome, disruption of one of them blocks all G protein-dependent pathways in LW6 mutant cells, which fail torespond to and to relay cAMP, to express aggregation spe-cific genes and to develop. In this strain only the rasGEFMupper transcript is detected during starvation, while thelower transcript fails to accumulate (Fig. 2B). This indi-cates that up-regulation of the lower transcript is undercontrol of the heterotrimeric G protein. Gene expressionwas also analyzed in the HSB1 and HSB50 mutants. HSB1is defective in cAMP relay, due to a temperature-sensitivemutation in the adapter protein PIA, which is essential foradenylyl cyclase activation [21,22]. HSB1 cells thus fail toaggregate but, in contrast to LW6, express at moderatelevel aggregation-specific genes and are sensitive to exoge-nous cAMP pulses. The HSB50 mutant undergoes chemo-taxis and aggregation, but is blocked at mound stage [23].gesting that transcription of the lower transcript does notrequire cAMP relay, even though it may be enhanced bycAMP pulses, similarly to other aggregation-specific genes[24]. It is worth mentioning that the two transcripts mayarise from differential splicing or different degree of poly-adenylation. Screening 24 cDNA clones obtained by usingmRNA from 6-h starved cells resulted in a signle sizedcDNA to be present. Sequencing three such clones gaverise to a transcript containing all four exons. Assumingthis transcript to correspond to the most abundant mRNAspecies at this time point, namely the lower band, then weshould conclude that differential splicing is unlikely andthe upper band is the result of extensive polyadenylation.Additional experiments are required to confirm thisNorthern blot analysis of rasGEFM expression in parental and mutant strainsFigur  2Northern blot analysis of rasGEFM expression in parental and mutant strains. (A) Total RNA was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radi-olabelled rasGEFM specific probe (probe a) corresponding to bp 760–1518 of the cDNA clone and to the actin gene used as a loading control. (B) Total RNA was extracted from dif-ferent developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts-mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).Disruption of the rasGEFM geneFigure 3Disruption of the rasGEFM gene. (A) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with EcoRI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a (rasGEFM N-terminal fragment cor-responding to bp 0–617 of the cDNA clone), probe b (corre-sponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c (bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the paren-tal strain. In the rasGEFM null strain the central part of the gene (recognized by probe b), was replaced by the blasticidin cassette (recognized by probe c), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifi-cally by bsr (probe c) or probe b in HSB61 or AX2, respec-tively. Both AX2 and HSB61 genomic loci are recognized by the probe a. (B) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and devel-opment. The membrane was hybridized to the rasGEFM and to the actin gene.Page 4 of 18(page number not for citation purposes)In both mutants, the rasGEFM expression pattern was sim-ilar to that observed in the parental strain (Fig. 2B), sug-hypothesis and to understand this intriguing develop-mentally regulated changes in rasGEFM gene expression.BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43rasGEFM null mutant is delayed in the acquisition of aggregation competence and blocked at the aggregation stageTo gain insight into the function of rasGEFM, the gene wasinactivated by homologous recombination. Disruptionwas confirmed by Southern and Northern blot analysis ofthe mutant strain, named HSB61 (Fig. 3). HSB61 cellsgrew normally both in axenic medium, or on bacteriallawn, but were defective in development. In contrast tothe parental cells (Fig. 4A), which form fruiting bodiesafter about 24 hours of starvation, HSB61 cells formedsmall, flattened aggregates, unable to develop into tippedtight mounds (Fig. 4B). Aggregate formation was density-dependent. If cells were plated on non-nutrient agar atconcentration of 1 or 2 × 105/cm2 no aggregates wereformed, whereas control cells formed aggregates. At 1 ×106 cells per cm2 the mutant cells formed small aggregates,similar to those shown in Fig. 4B, but many cells failed toaggregates. Treating cells with cAMP pulses rescued in partthe mutant phenotype; if aggregates, formed in suspen-sion after 6–8 hours pulses, were transferred on agar, theycontinued to develop and to form small fruiting bodies,although many cells failed to aggregate (Fig. 4C). We havefailed to observe cells forming streams, even in 6 to 8hours pulsed cells, although elongated cells very near toaggregates are found, suggesting that some short-rangechemotaxis may occur.The finding that HSB61 formed flat aggregates promptedus to test whether the mutant was defective in cell-celladhesion or chemotaxis. Intercellular adhesion in Dictyos-telium is developmentally regulated, as growth-phase cellsare weakly adhesive, and this adhesion is completelyblocked by EDTA. During the first 5–6 hours of develop-ment, cells express the adhesion glycoprotein csA on thecell surface, which is responsible for an EDTA-resistantform of adhesion (for a review see [25]), and at least inpart, for post-aggregative pattern formation [26]. Wetested the ability of HSB61 cells to develop EDTA-resistantadhesion over the first 8 hours of development. As shownin Fig. 5, HSB61 cells exhibited a delay of 5 to 6 hours inthe appearance of EDTA-resistant adhesiveness comparedto the parental strain. Cell treatment with cAMP pulses,which are known to stimulate expression of csA as well asseveral other aggregation-specific genes [19], acceleratedacquisition of EDTA-stable adhesion, though in contrastto the parental strain the mutant cells appeared to beDevelopmental phenotypes of (A) wild-type or (B, C) rasGEFM null cellsFigure 4Developmental phenotypes of (A) wild-type or (B, C) rasGEFM null cells. (A, B) AX2 or HSB61 cells were plated at the beginning of starvation at a concentration of 1 × 107cell/ml on non-nutrient agar (approx. 6.5 × 105 cell/cm2). (C) HSB61 cells were pulsed with cAMP for 10 hours before plated on agar. The final phenotype after 24 hours is shown.Appearance of EDTA-stable contacts in (open symbols) untr ted and (closed sym ols) cAMP treated cellsFigure 5Appearance of EDTA-stable contacts in (open sym-bols) untreated and (closed symbols) cAMP treated cells. Cells of (squares) AX2 or (triangles) HSB61 were devel-oped in suspension. At the developmental time indicated in the abscissa cells were taken, incubated with 10 mM EDTA and cell adhesion measured in the agglutinometer of Beug and Gerisch, as described in Methods. A representative Page 5 of 18(page number not for citation purposes)refractory to the pulses for at least 4 hours (Fig. 5).experiment is shown.BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43To examine whether rasGEFM null cells were impaired intheir ability to chemotax towards cAMP, a micropipette-based assay was used. HSB61 cells resulted stronglyimpaired in chemotaxis, as will be described furtherbelow, but their behaviour improved significantly whenpulsed with cAMP. Taken together these results suggestthat HSB61 cells fail to aggregate properly, due toimpaired periodic cAMP signalling, which is required foroptimal expression of aggregation-stage specific genes.To confirm this hypothesis, we followed the expression ofthree such genes, namely carA, acaA and csA. CarA andacaA encode the cAMP receptor cAR1 and adenylyl cyclaseA, respectively, whereas csA is, as mentioned above, themutant compared to the parental strain. The peak ofexpression for all three genes was reached in the parentalstrain between 4 and 6 hours of development followed bydown-regulation as cells undergo aggregation. Pulsingwith cAMP accelerated the kinetics of their expression,leading to a higher mRNA accumulation rate (Fig. 6 upperpanel). In the rasGEFM mutant, mRNA expression wasdelayed and down-regulation was not detected even after12 hours starvation. Cyclic AMP pulses elicited a stimula-tory effect, though not as efficient as in the parental strain(Fig. 6 bottom panel).Cyclic AMP receptor and G protein dependent activation of adenylyl and guanylyl cyclases is altered in rasGEFM null cellsThe finding that the HSB61 mutant was delayed in theacquisition of the aggregation competence but partiallyresponded to cAMP pulses suggested that the RasGEFMEarly developmental gene expression in AX2 and HSB61 cellsFigure 6Early developmental gene expression in AX2 and HSB61 cells. Total RNA was extracted from cells pulsed (+ cAMP) or not (- cAMP) in suspension for the time indicated. After electrophoresis and transfer, the membranes were hybridized with radiolabelled acaA, carA, csA. Actin was used as control.Cyclic AMP accumulation in AX2 and HSB61 during develop-ment in shaken suspensionFigure 7Cyclic AMP accumulation in AX2 and HSB61 during development in shaken suspension. (Squares) AX2 or (triangles) HSB61 cells were incubated in suspension for the time indicated in the abscissa. (A) control cells or (B) cells treated with cAMP pulses every 6 minutes. At the time indi-cated, cell aliquots were quenched with perchloric acid, neu-tralised with KOH, and total cAMP in the samples was determined by radioimmunoassay as described in Material and Methods.Page 6 of 18(page number not for citation purposes)gene for the cell adhesion glycoprotein csA. Expression ofacaA, csA, and to a lesser extent carA, was deranged in theprotein might be involved in cAMP receptor-mediated sig-naling. We therefore monitored cAMP accumulation dur-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43ing the early hours of development and tested adenylyland guanylyl cyclase activities in vitro.In AX2 cells, the basal cAMP is low at the beginning ofdevelopment and rises sharply at around 4 hours of devel-opment, reaching a maximum by around 7–10 hours,after which it decreases (Fig. 7A) [19,24]. In HSB61 cells,cAMP started to accumulate with a delay of 2 hours andincreased at a very low rate, reaching at 10 hours of starva-tion less than half the maximal concentration of the wildtype (Fig. 7A). Cell treatment with cAMP pulses acceler-ated of two hours endogenous cAMP accumulation bothin wild type and the mutant, restoring almost normal lev-els of cAMP in the latter (Fig. 7B).Cyclic AMP accumulation in the pre-aggregative andaggregation stage results from the periodic activation ofadenylyl cyclase, which is stimulated in autocrine andparacrine loops by cAMP. This leads to both increasedaccumulation of the enzyme and oscillatory stimulationof its activity, with a period of about 6 minutes [19]. Exog-enously supplied cAMP pulses mimic the endogenousoscillations of cAMP and give insight on potential defectsin cAMP relay or other cAMP induced responses in mutantcells. We investigated cAMP and cGMP changes in HSB61cells during a period of cAMP pulses. As shown in Fig. 8A,in response to a cAMP pulse, 5 hours starved and cAMP-treated HSB61 cells displayed a dramatically reducedincrease in cAMP compared to AX2 cells. In contrast thecGMP peak in the mutant was about twice that observedin the parental strain (Fig. 8B). Remarkably, when cellswere assayed after 9 hours pulsing, cGMP peaked 5 to 10fold higher compared to AX2 cells (Fig. 8C), while thecAMP level increased slightly compared to t5 mutant cells(data not shown). The rasGEFM null mutant, therefore,displays significantly increased cGMP and reduced cAMPresponses in the pre-aggregative stage and during aggrega-tion compared to the parental strain. The HSB61 develop-mental defects are supported by light scatteringmeasurements of cells in suspension, which showed thatthe mutant cells failed to undergo spontaneous light scat-tering oscillations. When pulsed with cAMP for severalhours, light scattering changes were induced, but theresponses were lower than in the wild type (data notshown).The finding of a reduced cAMP response in the mutantprompted us to assay adenylyl cyclase activity in celllysates upon stimulation of the cells with 2'-deoxy-cAMPor the slowly hydrolyzable GTP analog GTPγS. The assaywas done with mutant cells pulsed with cAMP for 7 hours,since after such treatment the mutant cells accumulatedcAMP to levels comparable to 5-h treated control cells, asCyclic AMP stimulation of (A) adenylyl and (B, C) guanylyl cyclase activityFigur  8Cyclic AMP stimulation of (A) adenylyl and (B, C) guanylyl cyclase activity. (Squares) AX2 or (triangles) HSB61 cells were treated with cAMP pulses for (A, B) 5 or (C) 9 hours. In conjunction with a cAMP pulse (arrows), sam-ples were taken at the time indicated in the abscissa for determining total concentration of (A) cAMP or (B, C) cGMP. Cyclic-nucleotides were measured using the radioimmu-noassay kit as described in the section Material and Methods. Representative experiments are shown.Page 7 of 18(page number not for citation purposes)shown in Fig. 7B. GTPγS stimulated adenylyl cyclase activ-ity about 16 and 13 fold in AX2 and HSB61 cells, respec-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43tively (Fig. 9). In contrast, stimulation with 2'-deoxy-cAMP increased the level of cAMP 4 fold in the mutantcompared to 44 fold for AX2 cells (Fig. 9). The finding thatGTPγS stimulated adenylyl cyclase to about the same levelin both strains suggests that basal adenylyl cyclase activityis roughly comparable in mutant and parental strain, andthis was confirmed by assaying adenylyl cyclase activity inthe presence of Mn2+ (Fig. 9). The strongly reduced adeny-lyl cyclase activation by 2'-deoxy-cAMP in the mutantcould be due to a lower number of cAMP receptorsexpressed on the cell surface. To exclude this we per-formed cAMP binding experiments in both cell lines AX2and HSB61 pulsed for 5 and 7 hours, respectively. Thetotal amount of cAMP receptors was comparable, as bothmaximum amount of ligand bound to the receptor(Bmax), and dissociation constant (Kd) were in the samerange in wild type and mutant cells (Fig. 10A).These data strengthen the hypothesis that the stronglyreduced adenylyl cyclase activation in HSB61 cells is Gprotein independent, and suggest that Ras GEFM may belocated upstream of the heterotrimeric G protein anddownstream of the cAMP receptor. If this hypothesis iscorrect, cAMP binding to membranes upon treatmentwith GTPγS should be altered in the mutant compared tothe wild type, since the affinity of cAMP receptors differswhen they are complexed with G proteins [27]. Consistentwith this hypothesis, cAMP binding to HSB61 membraneswas not affected by GTPγS, whereas it was inhibited cAMPbinding approximately 50% in wild type membranes (Fig.10B).Taken together, these results lead us to conclude that RasGEFM very likely acts between the cAMP receptor and theheterotrimeric G protein.RasGEFM is not the activator of RasC or RasGTwo previously characterized Ras proteins, RasC andRasG, have been shown to be involved in cAMP mediatedsignalling events [28-30]. Therefore, it was tempting tohypothesize that RasGEFM could function as the putativeexchange factor for either of these Ras proteins. ActivatedRas can be measured by using a Ras Binding Domain(RBD) to affinity purify Ras proteins. Given that the RBD-Ras interaction is dependent on Ras being in a GTP boundform, one can selectively measure activated Ras from cel-GTPγS and 2'-deoxy-cAMP induced adenylyl cyclase activa-tionFigure 9GTPγS and 2'-deoxy-cAMP induced adenylyl cyclase activation. AX2 or HSB61 cells were pulsed with cAMP for 5 or 7 hours, respectively. Cell lysates were prepared in the presence of (grey bars) GTPγS, (black bars) 2'-deoxy-cAMP, (black stripes) MnCl2 and assayed for adenylyl cyclase activity. Plotted values were normalized relative to the (open bars) unstimulated activity obtained in the absence of GTPγS or 2'-deoxy-cAMP (0.7 pmol/mg/min and 0.6 pmol/mg/min for AX2 and HSB61 respectively). Values for AX2 and HSB61 cell lysates are the means ± sd of two indipendent experi-ments run in duplicate.RasC and RasG activation in HSB61 mutantFigure 11RasC and RasG activation in HSB61 mutant. AX2 or HSB61 cells were pulsed with cAMP for the time indicated, concen-trated ten times, treated with cAMP and immediately lysed. The lysates were incubated with GST-Byr2 (RBD) as described in Material and Methods. The precipitate was subjected to electrophoresis and Western blot and hybridized with antibodies against RasC or RasG. Time (in seconds) after cAMP treatment is indicated. Total RasC and RasG in cell lysates from AX2 or Page 8 of 18(page number not for citation purposes)HSB61 are shown on the right.BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43lular lysates. An assay has recently been described for Dic-tyostelium Ras proteins employing the RBD from S. pombeByr2, and it has been shown that both RasC and RasG aretransiently activated by cAMP [30]. To determine whetherRasGEFM mediates this activation, we challenged 6 hourspulsed AX2 and HSB61 cells with cAMP and observed thekinetics of Ras activation (Fig. 11). While the levels ofboth activated RasC and RasG increased upon cAMP stim-ulation in HSB61, the maximum level of RasG-GTP wasgreatly reduced relative to AX2, whereas RasC activationwas largely unaffected in the mutant. This suggested thepossibility that RasGEFM may be partly responsible formediating the activation of RasG. However, when 10-hour pulsed cells were stimulated with cAMP, RasG acti-vation was restored. Interestingly, the total level of RasGremained at a very high level relative to AX2. This datashows that RasGEFM is unlikely to be an activator of RasCor RasG, and the partial loss of RasG activation seen in 6hours cells may be a secondary effect of reduced geneexpression. In addition, the finding that RasG is activatedafter prolonged cAMP treatment favours the notion that adevelopmentally regulated component, regulated bycAMP signalling, is directly or indirectly required for RasGactivation.Ca2+ influx in rasGEFM null mutant is reducedIn addition to the effects on cAMP and cGMP levels, thesecond messenger Ca2+ is rapidly regulated by cAMP bind-ing to the receptor [31]. The finding that chemotaxis andcGMP response were altered in the mutant, prompted usto test chemoattractant-induced Ca2+ entry in HSB61 cells.Ca2+ entry is negatively regulated by cGMP [32], and bothCa2+ and cGMP have been implicated in regulating chem-otactic motility [33].The basal levels of intracellular Ca2+ concentration werecomparable in parental and mutant cells and elevation inresponse to a cAMP pulse was only slightly reduced in themutant (data not shown). Cyclic AMP-induced Ca2+influxdepends on the extracellular Ca2+concentration and onthe dose of the cAMP stimulus. The influx is increasedwith elevation of the concentration of both parameters[34]. Ca2+ influx is maximal in AX2 cells after 4 to 6 hoursof development and remains constant thereafter [34]. Wedetermined maximal Ca2+ influx in unpulsed and pulsedHSB61 cells. As shown in the saturation curve of Fig. 12A,left, maximal Ca2+ influx was reduced to about 50% of thecontrol in unpulsed HSB61 cells, but influx was restoredwhen the mutant cells were pulsed with cAMP (Fig. 12A,right). Surprisingly, both in unpulsed or cAMP pulsedHSB61 cells, the kinetics of single Ca2+response was asrapid as the wild type in terms of influx and efflux (Fig.12B, a-b). The increased concentration level of cGMP in2+ high to affect Ca2+ entry [27] or that cGMP has no effecton Ca2+ influx [35].As shown in Fig. 12Bd, AX2 cells at 8 hours of develop-ment, which corresponds to late aggregation stage, dis-played a rapid Ca2+ influx in response to cAMP, followedby a very slow efflux. Later on in development, no effluxcould be detected [34]. In HSB61 cells, even when pulsedfor as long as 12 hours, the kinetics of efflux was similarto 4–6 hours AX2 pulsed cells (Fig. 11B, b and c). Thus,these data further confirm that the mutant development isdelayed even after 12 hours pulsing with cAMP. In addi-tion, the finding that maximal Ca2+ entry in response tocAMP is reduced in the mutant may account, at least inpart, for the chemotactic defect of the mutant.Mutant HSB61 is impaired in chemotactic, but not in spontaneous cell motilityWhen cell motility was analysed, differences betweenmutant and parental strains were only detected duringchemotaxis. Spontaneous cell motility was indistinguish-able between both cell types. Directional cell migration inresponse to external chemoattractant gradients implies atleast three steps: a) sensing the chemoattractant, b) forma-tion of a leading front and c) cell polarization, with sup-pression of lateral pseudopods, followed by forwardmovement and uropod detachment [33].As depicted in Fig. 13 and additional file 1, aggregationcompetent wild type cells, when challenged with thecAMP-loaded micropipette, become highly polarized,with a clearly defined leading front and a posterior uro-pod, and rapidly move towards the cAMP source. In theirmovement, AX2 cells eventually adhere to each other intostreams, due to outward relay of the signal (Fig. 13, upperpanel). Five hours-starved HSB61 null cells behaved dif-ferently: they sensed the gradient and extended a mem-brane lamella towards the micropipette, but they alsoextended several lateral pseudopodia with high frequency,thus displaying a severe polarization defect. In contrast tothe parental strain, the mutant cells exhibited a rather flat-tened shape and an apparently increased cell-substratumadhesion. As a result, their chemotactic orientation andmotility were strongly reduced (Fig. 13, middle panel andadditional file 2). Pulsing with cAMP for at least 6–8hours partially rescued the mutant cell phenotype, in thatthese cells were now better polarized and displayed anorganized leading front, moving towards the capillary in away similar to AX2, though the cell population was some-what heterogeneous in that respect (Fig. 13, lower paneland additional file 3). The chemotactic speed of mutantcells changed from 1.43 ± 0.49 µm/min for untreated cellsto 6.2 ± 2.1 µm/min for cAMP pulsed cells. Both valuesPage 9 of 18(page number not for citation purposes)the HSB61 mutant did not affect the timing of Ca influx.This suggests that either cGMP level must be constantlywere below the 9.7 ± 1.81 µm/min observed for 5 hoursstarved AX2 cells.BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43Cyclic AMP pulses failed to induce cell streaming: theHSB61 cells chemotaxed towards the capillary mostly assingle cells, suggesting that spontaneous cAMP relay wasstill reduced in the mutant.Stimulation with chemoattractants causes polymerizationand reorganization of actin, and this has been correlatedwith the extension of new pseudopods during chemotaxis[36]. We investigated the levels of F-actin in HSB61 cellsfollowing cAMP pulses. Wild-type cells showed theexpected biphasic response with a sharp peak of F-actin atabout 5 seconds after stimulation and a lower secondpeak at about 50 seconds (Fig. 14A). A very low actinresponse was detected in HSB61 cells pulsed for 4 hoursthe intensity of actin response was similar to that of theparental strain (Fig. 14C).DiscussionIn this study we have reported the isolation and character-ization of a novel RasGEF, named RasGEFM, which isrequired for proper Dictyostelium development. RasGEFMis peculiar in that it contains 6 poly-proline stretches thatrepresent putative interacting sites for SH3, WW and/orEVH1 containing proteins [37], and a DEP1 motif.Disrupting rasGEFM results in cells to be blocked at theaggregation stage, forming rather small, flattened aggre-gates that fail to develop further. The RasGEFM-null phe-cAMP binding to the cell surface and its inhibition by GTPγSFigure 10cAMP binding to the cell surface and its inhibition by GTPγS. (A) Scatchard analysis of cAMP binding in AX2 and HSB61 cells pulsed with cAMP for 5 or 7 hours, respectively. The [3H] cAMP binding was determined over a range of 700–19700 nM cAMP by incubation for 5 min at 0°C. A fitted line, Bmax and Kd are shown in each panel. (B) Inhibitory effect of GTPγS (black bar) on the binding of cAMP to their cognate receptors. Crude membranes were incubated with [3H] cAMP in the absence or presence of GTPγS. Values are indicated as the percentage of cAMP binding in treated normalized to the untreated membranes (white bar). In wild type strain cAMP binding dropped from 10.4 ± 0.3 nM to 5.26 ± 0.04 nM for GTPγS untreated and treated membranes, respectively. HSB61 strain displayed comparable absolute values: 16.23 ± 0.25 and 16.00 ± 0.3 nM for GTPγS untreated and treated membranes, respectively. Means of three independent experiments, each run in dupli-cate, are shown.Page 10 of 18(page number not for citation purposes)(Fig. 14B), but when cells were pulsed for at least 6 hours notype could be partially rescued by pulsing cells withcAMP. Gene expression studies and functional assays indi-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43cate that the mutant phenotype is due to defective devel-opmental expression of cAMP relay and are consistentdisplayed by other mutants that are strictly defective inadenylyl cyclase activation, such as mutants in RasGEFACa2+ influx in HSB61 mutantFigure 12Ca2+ influx in HSB61 mutant. A) Dependence of cAMP-induced Ca2+-influx from [Ca2+]e measured in unpulsed and pulsed HSB61 cell suspensions. Dose-response curves are shown for (left) untreated cells and (right) cells treated with 20 nM cAMP pulses. In both cases, cells were tested after 4–5 hours starvation. White circles depict the HSB61 cells and black circles the AX2 cells. Data are presented for at least 4 independent experiments for each cell type. P values = 0.00068 and 0.8368 for left and right graphs, respectively (Wilcoxon test). B) cAMP-induced Ca2+-influx in HSB61 and wild-type cells. Single electrode recordings are shown for (a, b) mutant cells either (a) unpulsed or (b) pulsed overnight with 20 nM cAMP. For comparison (c, d) wild-type cells are shown after 5 hours of development in (c) and after 7.5 hours in (d). Addition of cAMP is indicated.Page 11 of 18(page number not for citation purposes)with a role of RasGEFM in regulating cAMP signalling. Themutant phenotype differs, however, from the phenotype[9], PIA [21,22] CRAC [38] and RasC [29]. PIA and CRACare cytosolic proteins that couple the G protein to adeny-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43lyl cyclase, and RasGEFA as well as RasC have been shownto also be essential for adenylyl cyclase activation. In con-trast to rasGEFM null cells, which form small flat aggre-gates, all these other mutants fail to aggregate, althoughthey respond to cAMP pulses. Their chemotactic motilityin response to cAMP diffusing from a microcapillary isalso much less impaired, and guanylyl cyclase activationis rather normal and not enhanced as in the rasGEFM nullmutant.These findings indicate that these mutants display differ-ent alterations in signal transduction downstream of thecAMP receptor. The RASGEFM protein, in contrast to Ras-GEFA, PIA and CRAC, appears to be located downstreamof the cAR1 receptor, but upstream of the G protein, thuspossibly affecting both G protein dependent and G pro-tein independent pathways.RasGEFM null cells have defects in both adenylyl cyclasefore, that RasGEFM would act upstream of RasC. How-ever, we now have direct evidence that RasGEFA directlyactivates RasC and is the only GEF responsible for the acti-vation of RasC (Kae et al, unpublished). Consistent withthis result, there was no major effect on the cAMP depend-ent activation of both RasC and RasG in the rasGEFM nullcells.Several attempts have been performed to try to rescue theHSB61 phenotype but so far none of them succeeded,although a RasGEFM GFP-fused protein of the correct sizewas expressed (data not shown). This observation, com-bined with the rather peculiar rasGEFM mRNA expres-sion, in which two transcripts are detected and regulatedindependently, suggests that the failed rescue might bedue to the necessity for the RasGEFM protein to beexpressed in a regulated way and not under the control ofa constitutive promoter, such as the actin 15 promoter.Experiments are in progress in this direction and they willChemotaxis of wild-type and HSB61 cellsFigure 13Chemotaxis of wild-type and HSB61 cells. Cells were developed in shaken suspension, either in the presence or absence of exogenous cAMP pulses, plated on coverslips and tested for chemotaxis towards a microcapillary diffusing cAMP. Upper and middle panels show AX2 or HSB61 cells starved for 5 hours, bottom panel shows HSB61 cells treated with cAMP pulses for 10 hours (see additional files 1, 2, 3). Higher magnifications of each cell sample are shown on the right. Numbers: time in minutes, starting after positioning of the microcapillary (0' time).Page 12 of 18(page number not for citation purposes)activation and in chemotaxis, characteristics similar tothose of rasC null cells. It might have been expected, there-be crucial to confirm that the phenotype is due to directdisruption of the rasGEFM gene and not to additional det-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43rimental effects of the transformation. The possibility of asecond random mutation, which independently of ras-GEFM disruption could be responsible for the observedphenotype, cannot be excluded but it seems unlikely tous. The GTPγS inhibition of cAMP binding, together withthe stimulating effects of GTPγS on adenylyl cyclase, andthe rescuing effect of cAMP, clearly locate the mutantdefect upstream of the G protein and suggest a singleevent to be involved.When rasGEFM null cells are challenged with a cAMPfilled micropipette, the chemotactic response is impaired.Apparently the cells sense the gradient but they seem inca-pable of organizing a leading front. Moreover, several lat-eral pseudopodia are continuously extended andretracted. As a consequence, the cell fails to polarize,maintains a rather flattened shape and moves "spasti-cally" towards the micropipette. If mutant cells are treatedwith cAMP pulses for at least 6 to 8 hours, chemotactic ori-entation and speed are improved, and many cells becomeelongated and move faster towards the micropipette.Formation of a leading front, acquisition of polarity andsuppression of lateral pseudopodia are processes charac-terized by the redistribution of cytoskeletal components,with F-actin and numerous actin-binding proteins beingenriched at the front and myosin II assembled in fila-ments at the back of cells [33]. These processes are regu-lated by the balanced action of several sub-cellularcomponents, including second messengers (e.g. Ca2+,cGMP) and proteins such as PI3K and PTEN [39]. A mech-anism by which localized Ras activation mediates leadingedge formation through activation of PI3K and other Raseffectors required for chemotaxis has been recently pro-posed for Dictyostelium cells by Sasaki et al. [28].Cyclic AMP elicits in the mutant a biphasic actin polymer-ization response comparable to the parental strain, butthe absolute peak values are strongly reduced. Strikingly,when mutant cells are pulsed for additional 2–4 hours theactin response is comparable to that of the wild type.These results suggest to us that RasGEFM is not directlyinvolved in mediating a putative Ras-induced actinpolymerization. They are instead consistent with thenotion that a developmentally regulated component,required for proper actin recruitment in response to che-moattractant, is absent or expressed under a thresholdlevel in the mutant and is induced by prolonged cAMPtreatment.Taken together our findings support a role for RasGEF Min developmental acquisition of chemotactic efficiencyand aggregation competence. The proposed localizationsociating components between G protein coupled recep-tors and the G protein or molecular switches acting in a Gprotein independent way. A physical interaction betweenβ 1 adrenergic receptors and a RasGEF has been recentlyreported in mammalian cells [40].A peculiarity of the RasGEFM null mutant is the elevatedcyclic GMP response to cAMP stimulation both at earlyand late starvation times, which suggests a role for Ras-GEFM as negative regulator of cAMP receptor-inducedguanylyl cyclase activation. Cyclic GMP accumulation hasbeen proposed to be regulated by adaptation [41] and toinhibit pseudopodia formation at the back of the cell byinducing myosin filament formation in the cell cortex[42-45]. Myosin filament formation at the presumptiveleading front would be counteracted by myosin phospho-rylation due to myosin heavy chain kinase A (MHCK-A),which is selectively recruited at the leading front [45]. Asa result, a high cGMP concentration leads to improvedorientation in a chemoattractant gradient, as shown inmutant cells lacking cGMP phosphodiesterase [46]. Ras-GEFM null cells, challenged with cAMP diffusing from amicrocapillary, show, however, reduced orientation andincreased lateral pseudopodia, despite a stronger cGMPresponse to cAMP. It may be argued that cGMP is rapidlyhydrolysed in the HSB61 mutant, in contrast to the cGMPphosphodiesterase null cells. However, if cGMP is down-regulated by adaptation, it must be assumed that as longas cells are exposed to a chemoattractant gradient, asoccurs when they are stimulated with cAMP diffusingfrom a microcapillary, adaptation of guanylyl cyclase isswitched off [41], and this should lead in RasGEF M nullcells to constantly higher levels of cGMP. Why is thenchemotactic orientation reduced in the mutant?A possible explanation for this discrepancy is offered bythe reduced actin response to chemoattractant. We havesuggested that a developmentally regulated component,which is required for actin recruitment at the presumptiveleading front, is lacking/down-regulated in the mutant.Strongly reduced actin polymerization at the front wouldresult in impaired translocation of myosin heavy chainkinase A (MHCK-A), which has been shown to require F-actin binding [47]. As a consequence, a high cGMP levelin the mutant, as in wild type cells or PDE null mutants,would induce myosin filaments all over the cell, consist-ent with its proposed role as global inhibitor [33,45], butmyosin filaments in the front would not be dissociated byMHCK-A, due to its impaired recruitment. Thus we pro-pose that in the RasGEFM null mutant chemotaxis isinhibited due firstly to altered F-actin polymerization atthe presumptive leading front and secondarily toimpaired recruitment of MHCK-A.Page 13 of 18(page number not for citation purposes)of Ras GEFM downstream the cAMP receptor lead us tosuggest that Ras GEFM and its cognate Ras may act as dis-BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43ConclusionAll the defects observed in the rasGEFM null mutant canbe explained by assuming a modulatory role of the Ras-GEFM close to the cAMP receptor, which regulates guany-lyl and adenylyl cyclases. RasGEFM appears to act as anegative regulator of guanylyl cyclase and a positive regu-lator of adenylyl cyclase. Currently we have no obviousexplanation for this opposite activity. It must be kept inmind, however, that in contrast to adenylyl cyclase, whichis stimulated by the Gβ subunit of the heterotrimeric Gprotein, receptor-dependent stimulation of guanylylcyclase is more complex and less well understood. Severallines of evidence suggest a role for small GTPases in itsactivation, independently and in addition to the heterot-rimeric G protein [48-50]. If RasGEFM and its putative Rastarget act as receptor-linked molecular switches, this maylead to differential, albeit opposite, effects on heterot-rimeric G-protein-dependent or independent pathways.Identifying the putative RasGEFM regulated Ras may helpin understanding these complex transduction pathways.MethodsCell cultures, growth, and developmental conditionsWild type strain AX2-214 and rasGEFM null mutant,referred as HSB61, were grown either in liquid nutrientmedium at 23°C under shaking at 150 rpm [51] or onnutrient agar plates with Escherichia coli B/2 [52]. Whencultured in liquid medium, HSB61 cells were supple-mented with blasticidin (ICN) at a final concentration of10 µg/ml. For development on solid substratum, cellswere grown to a density of 2–3 × 106 cell/ml, washed threetimes in 0.017 M Na+/K+ Soerensen phosphate buffer, pH6.0 and, once deposited on non nutrient 1.5% (w/v) agarplates, allowed to develop at 23°C. For development insuspension, cells were incubated at a concentration of 1 ×107 cell/ml in Soerensen phosphate buffer. For cAMPtreatment, pulses of 20 nM cAMP were applied every 6minutes using a Braun perfusor VI equipped with 10 ml-syringe [53].Measurement of EDTA-stable contactsEDTA-stable contacts were measured as described byusing the agglutinometer of Beug and Gerisch [52].Chemotaxis assayChemotaxis was studied by using the microcapillary assay[54]. Briefly, cells were seeded onto 35 mm glass basedishes (Iwaki) at a density of approximately 1 × 105/cm2and local stimulation of chemotaxis was obtained by pas-sive diffusion of cAMP from a microcapillary (Femtotips1,Eppendorf), filled with a 1.0 mM solution of cAMP. Themicrocapillary was positioned with an automated Zeissmicromanipulator and cells observed either with a 20× orActin polymerization assay in response to chemoattractant stimulationFigure 14Actin polymerization assay in response to chemoat-tractant stimulation. The cells concentrated at 2 × 107/ml were stimulated with 1.0 µm cAMP (time 0) and the F-actin formation measured with the phalloidin binding assay as described in Material and Methods. Starving (A) AX2 and (B, C) HSB61 cells were treated with cAMP pulses and tested for F-actin polymerization after (A, B) 4 or (C) 6 hours of starva-tion.Page 14 of 18(page number not for citation purposes)with a Neofluar 100x/1.3 oil immersion objective,equipped with DIC filter. Images were captured at anBMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43interval of 0.66 sec. and recorded in a Panasonic videore-corder (AG-TL700) with a ZVS-47DE camera (Zeiss)mounted on a microscope (Zeiss, Axiovert HAL100). Therecorded time-lapse movies were transferred to a compu-ter using USB Instant Video package (ADS Technologies).Actin polymerization assayActin polymerization assays were carried out as previouslydescribed [36,55]. Briefly, cells were starved at 2 ×107cells/ml for 4 and 6 hours and pulsed with 1 µmcAMP. At the indicated time points, 100 µl samples weretaken and transferred to 1 ml of actin buffer (20 mMK2PO4, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, 3.7%formaldehyde, 0.1% Triton X-100, 0.25 µM TRITC-phal-loidin, pH6.8). After shaking for 1 hour at room tempera-ture, samples were centrifuged for 10 minutes at 16000 gand the resulting pellet was resuspended in 1 ml metha-nol. After shaking overnight, the amount of F-actin wasdetermined by measuring the fluorescence with a fluorim-eter (Kontron SFM 25). Setting: excitation wavelength 540nm, emission 570 nm.Measurement of cAMP-induced Ca2+-influxNet Ca2+-influx after agonist stimulation was done asdescribed previously [34,56].Cells were developed in suspension as described above.For accelerating developmental gene expression cells werepulsed with 20 nM cAMP every 6 minutes, overnight incase of HSB61 cells, and for 2 hours in case of wild-typecells. At appropriate time points the cells were washed innominally Ca2+ free tricine buffer (tricine pH 7.0, supple-mented with 5 mM KCl) and resuspended at a cell densityof 5 × 107 cells/ml. The cell suspension was then stimu-lated with cAMP and Ca2+-influx measured with a Ca2+-sensitive electrode (Möller) and a voltmeter (Metrohm).Statistical analysis was performed with "Wilcoxon test".Light scattering measurements of cells in suspension weredone has described by Gerisch and Hess [57]. The cell sus-pension (2 × 107cell/ml) was aerated in a cuvette andextinction was concomitantly monitored at 500 nm in aZeiss PM6 spectrophotometer.In vitro stimulation of adenylyl cyclaseAdenylyl cyclase in Dictyostelium lysates was assayed in thepresence of 2'-deoxy-cAMP or GTPγS as described by Lillyand Devreotes [58]. Briefly starving cells were treated with20 nM cAMP pulses every 6 min for different hours. Atotal of 1 × 108 cells were pelleted and resuspended in 1ml of Soerensen phosphate buffer. An equal volume of icecold lysis buffer containing either 4 mM MgCl2 or 4 mMMnCl2, 20 mM Tris pH 8.0 was added. Cells were lysed bypassage through a 3-µm pore size Nucleopore membrane40 µl aliquot of cell lysate was added to a 40 µl assay mix(20 mM DTT, 1 mM ATP, 2 mM MgCl2 or 2 mM MnCl2 in10 mM Tris pH 8.0, 0.4 mM IBMX) and incubated at 20°Cfor 5 min.The reaction was stopped, by adding 40 µl 0.1 M EDTA pH8.0 and boiling the sample for 2 min. The total concentra-tion of cAMP in the samples was determined by using the"Biotrak cAMP assay Kit" according to manufacturer'sinstructions (Amersham Pharmacia Biotech).cAMP binding assaysBinding of cAMP to cell surface receptors was determinedas previously described by Van Haastert [59]. Briefly, wild-type and mutant cells were starved by shaking in Soer-ensen phosphate buffer, without or with cAMP pulses for5 or 8 hours respectively, washed and resuspended at adensity of 1 × 108 cell/ml.Aliquots of 80 µl of cells were incubated with a radioactivebinding mixture, containing 300 nM of [3H]cAMP (Amer-sham Pharmacia Biotech), 50 mM dithiothreitol in 90%saturated ammonium sulphate, and a variety of cAMPconcentration ranging between 700 to 19700 nM. Specificbinding was obtained by subtracting non specific bindingdetermined in the presence of 1 mM cAMP.After 5 min. incubation at 0°C, cells were collected bycentrifugation at 14000 × g for 2 min, the pellet resus-pended in 100 µl of 0.1 M acetic acid and dissolved in 1.3ml scintillation fluid. Scatchard plots of cAMP bindingwere done with GraphPad software (GraphPad Inc.).GTP-inhibition of cAMP binding to plasma membranesThe assay was performed as described earlier [27]. Brieflly,GTP-inhibition of cAMP binding was measured in a totalvolume of 100 µl containing PB (10 mM KH2PO4/Na2HPO4 pH 6.5), 5 nM [3H] cAMP, 10 mM dithiothrei-tol, GTPγS (300 µM when present) and 70 µl membranes.Samples were incubated 5 min. at 0°C, centrifuged for 2min. at 14000 × g, the supernatant was aspirated and thepellet dissolved in 100 µl acetic acid. Radioactivity wasdetermined after the addition of 1.3 ml of liquid scintilla-tion.Cyclic AMP and cGMP determinationFor the determination of cyclic nucleotides, cells aliquotswere taken at different time points before and after acAMP pulse, and quenched with 1 vol of 2 N perchloricacid [24]. After centrifugation, neutralization of the super-natant with potassium carbonate, and acetylation, theconcentration of cAMP and cGMP in the extract was meas-ured using the 125I radioimmunoassay kit according toPage 15 of 18(page number not for citation purposes)in the absence or presence of 30 µM GTPγS or 50 µM 2'-deoxy-cAMP and the lysates incubated on ice for 5 min. Amanufacturer's instructions (Amersham Pharmacia Bio-tech).BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43RBD binding assayThe RBD binding assay was performed as described else-where [30]. Briefly, 6 h or 10 h pulsed cells were washedtwice and resuspended at 5 × 107 cell/ml in Soerensenphosphate buffer. Cells were stimulated with 200 nMcAMP, aliquots (0.5 ml) were selected at the indicatedtime points and lysed in an equal volume of ice-cold 2×HK-LB (20 mM sodium phosphate pH 7.2, 2% Triton X-100, 20% glycerol, 300 mM NaCl, 20 mM MgCl2, 2 mMEDTA, 2 mM Na3VO4, 10 mM NaF, with protease inhibi-tor Roche), and incubated on ice for 5 min. The lysateswere cleared by centrifugation for 10 min and proteinconcentrations were determined using DC Protein Assay(Bio-Rad). A 0.8 mg portion of protein lysates was incu-bated with 100 µg of GST-Byr2 (RBD) and the mixturewas incubated at 4°C for 1 h. Beads were harvested bycentrifugation and washed three times in 1× HK-LB. A vol-ume of 40 µl of 1× SDS gel loading buffer was added tothe pellet beads and the mixture was boiled for 5 min.Samples were fractionated by SDS-PAGE, blotted, blockedand probed with RasC or RasG antibody. Bands weredetermined by enhanced chemiluminescence reaction(Amersham Pharmacia Biotech).Molecular cloning and sequence analysisThe rasGEFM gene was isolated using PCR based method.Through the sequence derived from DNA database screen-ing [13,14] a pair of primers was designed (5'-ATGAT-GAATGAAGTTTCTTCAAATTC-3' and 5'-CCATCGATAATTATCTAAATAATGGATTTGA-3') and usedfor PCR amplification. As template, cDNA isolated with"First strand cDNA synthesis kit" (Amersham PharmaciaBiotech), from RNA of cells developed for 5 hours onsolid substrata, was used. The DNA fragment, of approxi-mately 2.7 kb, was then ligated into "pGEM-T easy vector"(PROMEGA) and cloned into DH5α E. coli strain. PCRproducts were purified from gel with "High Pure PCRProduct Purification" kit (Roche) and verified by sequenc-ing.Construction of the D.d. rasGEFM null strain (HSB61)To construct the rasGEFM null strain the rasGEFM locuswas disrupted via homologous recombination. The blasti-cidin resistance gene (bsr), used as selectable marker, wasexcised from pUCBsr∆ Bam [60] with HindIII and XbaIand subsequently inserted into the JC2a86b07.s1 clone(Dictyostelium genome project) digested with HindIII andXbaI. Subsequently the N-terminal portion (from position0 to 617 bp) of the gene was ligated into KpnI site of theabove vector, using the "DNA ligation kit" (AmershamPharmacia Biotech). The vector, carrying the selectablemarker was electroporated [61] into parental strain andtransformed cells selected for blasticidin resistence. Resist-via Southern blot in order to identify Dictyostelium clonesin which the RasGEF M locus was disrupted.Southern and Northern hybridization analysisGenomic DNA was extracted and purified by CsCl gradi-ent centrifugation as previously described by Nellen, et al.[62], digested with EcoRI, run onto 0.8% agarose gel, blot-ted onto Hybond-N membrane (Amersham PharmaciaBiotech), and subjected to Southern assay [63]. The mem-brane was probed with a 800 bp RasGEF M cDNA specificprobe, corresponding to bp. 760–1518 of the cDNAclone, previously radiolabelled by the "Megaprimer™DNALabelling System" using [α32] dATP (Amersham).For Northern blots, total RNA was prepared using TRIZOLreagent (GIBCO) according to manifacturer's instructions.RNA was then resuspended in DEPC treated water, quan-tified, and 15 µg were size separated on 1.2% agarose gelin presence of formaldehyde. Equal loading of samples,was checked by probing membranes with the actin gene.The radiolabelled DNA fragments used as probes were asfollow: car1, csA, acaA (fragment from 2.7 kb to 4.2 kb ofcDNA clone), RasGEF M. After being hybridised with thefirst cDNA probe, Northern blots were stripped with 0.1%SDS in boiling water and then re-hybridised with a secondprobe.Authors' contributionsM. A. and E. B. carried most of the experiments and wereinvolved in drafting the manuscript. D. L. was responsiblefor light scattering oscillations and Ca2+ analysis, H. K.and G. W. for pull down experiments with RasC and RasG.S. B. supervised the work and revised the manuscript, withcontributions by all authors.Additional materialAdditional File 1Chemotaxis of AX2 after 5 hours of starvation.mov: 6.5 MB. Cells were seeded onto 35 mm/glass base dish (Iwaki) and subjected to a cAMP filled micropipette assay, as described in Material and Methods. Images were recorded in a Panasonic videorecorder (AG-TL700) with a ZVS-47 DE camera (Zeiss) mounted on an Axiovert HAL100, using Neofluar 100X/1.3 oil immersion objective and DIC filter. The recorded time-lapse movie was transferred to a computer using USB Istant Video (ADS Tech-nologies).Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2121-6-43-S1.mov]Additional File 2Chemotaxis of HSB61 after 5 hours of starvation.mov: 6.9 MBClick here for file[http://www.biomedcentral.com/content/supplementary/1471-2121-6-43-S2.mov]Page 16 of 18(page number not for citation purposes)ant cells were cloned, and clones subsequently screenedBMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/43AcknowledgementsWe thank Barbara Peracino for technical help and D. Malchow for helpful suggestions. This work was supported by funds of MIUR (cofin 2002) to S.B. and local funds of the University of Turin to E.B.References1. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ:Increasing complexity of Ras signaling.  Oncogene 1998, 17(11Reviews):1395-1413.2. Lim CJ, Spiegelman GB, Weeks G: Cytoskeletal regulation byDictyostelium Ras subfamily proteins.  Journal of Muscle Research& Cell Motility 2002, 23(7–8):729-736.3. Takai Y, Sasaki T, Matozaki T: Small GTP-binding proteins.  Phys-iological Reviews 2001, 81(1):153-208.4. Bourne HR, Sanders DA, McCormick F: The GTPase superfamily:conserved structure and molecular mechanism.  Nature 1991,349(6305):117-127.5. Boguski MS, McCormick F: Proteins regulating Ras and its rela-tives.  Nature 1993, 366(6456):643-654.6. Wilkins A, Insall RH: Small GTPases in Dictyostelium: lessonsfrom a social amoeba.  Trends Genet 2001, 17:41-48.7. Broek D, Toda T, Michaeli T, Levin L, Birchmeier C, Zoller M, PowersS, Wigler M: The S. cerevisiae CDC25 gene product regulatesthe RAS/adenylate cyclase pathway.  Cell 1987, 48(5):789-799.8. Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM: Ras1and a putative guanine nucleotide exchange factor performcrucial steps in signaling by the sevenless protein tyrosinekinase.  Cell 1991, 67(4):701-716.9. Insall RH, Borleis J, Devreotes PN: The aimless RasGEF isrequired for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium.  Current Biology1996, 6(6):719-729.10. Wilkins A, Chubb JR, Insall RH: A novel Dictyostelium RasGEF isrequired for normal endocytosis, cell motility and multicel-lular development.  Current Biology 2000, 10(22):1427-1437.11. Goldberg JM, Bosgraaf L, Van Haastert PJ, Smith JL: Identification offour candidate cGMP targets in Dictyostelium.  Proceedings ofthe National Academy of Sciences of the United States of America 2002,99(10):6749-6754.12. Bosgraaf L, Russcher H, Smith JL, Wessels D, Soll DR, van HaastertPJM: A novel cGMP signalling pathway mediating myosinphosphorylation and chemotaxis in Dictyostelium.  EMBO J2002, 21:4560-4570.13. Dictyostelium genome project database:   [http://genome.imb-jena.de/Dictyostelium]14. Dictyostelium genome project database:   [http://seqtool.sdsc.edu/GCI/BW.cgi]15. Macias MJ, Wiesner S, Sudol M: WW and SH3 domains, two dif-ferent scaffolds to recognize proline-rich ligands.  FEBS Letters2002, 513(1):30-37.16. Lai CCBM, Broek D, Powers S: Influence of Guanine Nucleotideson complex formation between Ras AND CDC25 proteins.Molecular and Cellular Biology 1993, 13(3):1345-1352.17. Ponting CP, Bork P: Pleckstrin's repeat performance: a noveldomain in G-protein signaling?  Trends in Biochemical Sciences1996, 21(7):245-246.18. Wong HC, Mao J, Nguyen JT, Srinivas S, Zhang W, Liu B, Li L, Wu D,Zheng J: Structural basis of the recognition of the dishevelledDEP domain in the Wnt signaling pathway.  Nature Structural19. Gerisch G: Cyclic AMP and other signals controlling celldevelopment and differentiation in Dictyostelium.  Annu RevBiochem 1987, 56:853-879.20. Lilly P, Wu L, Welker DL, Devreotes PN: A G-protein beta-subu-nit is essential for Dictyostelium development.  Genes & Devel-opment 1993, 7(6):986-995.21. Chen MY, Long Y, Devreotes PN: A novel cytosolic regulator,Pianissimo, is required for chemoattractant receptor and Gprotein-mediated activation of the 12 transmembranedomain adenylyl cyclase in Dictyostelium.  Genes Devel 1997,11:3218-3231.22. Pergolizzi B, Peracino B, Silverman J, Ceccarelli A, Noegel A, Devre-otes P, Bozzaro S: Temperature-sensitive inhibition of devel-opment in Dictyostelium due to a point mutation in the piaAgene.  Developmental Biology 2002, 251(1):18-26.23. Ceccarelli A, Bozzaro S: Selection of mutants defective in bind-ing to immobilized carbohydrates in Dictyostelium discoi-deum.  Anim Biol 1992, 1:59-68.24. Bozzaro S, Hagmann J, Noegel A, Westphal M, Calautti E, Bogliolo E:Cell differentiation in the absence of intracellular and extra-cellular cyclic AMP pulses in Dictyostelium discoideum.  DevBiol 1987, 123:540-548.25. Bozzaro S, Ponte E: Cell adhesion in the life cycle of Dictyostel-ium.  Experientia 1995, 51:1175-1188.26. Queller DC, Ponte E, Bozzaro S, Strassmann JE: Single-gene green-beard effects in the social amoeba Dictyostelium discoi-deum.  Science 2003, 299:105-106.27. Van Haastert PJM: Alteration of Receptor/G-protein Interac-tion by Putative Endogenous Protein Kinase Activity in Dic-tyostelium discoideum Membranes.  J Biol Chem 1987,262(7):3239-3243.28. Sasaki AT, Chun C, Takeda K, Firtel RA: Localized Ras signalingat the leading edge regulates PI3K, cell polarity, and direc-tional cell movement.  Journal of Cell Biology 2004, 167(3):505-518.29. Lim CJ, Spiegelman GB, Weeks G: RasC is required for optimalactivation of adenylyl cyclase and Akt/PKB during aggrega-tion.  EMBO Journal 2001, 20(16):4490-4499.30. Kae H, Lim CJ, Spiegelman GB, Weeks G: Chemoattractant-induced Ras activation during Dictyostelium aggregation.EMBO Reports 2004, 5(6):602-606.31. Schaloske RH, Lusche DF, Bezares-Roder K, Happle K, Malchow D,Schlatterer C: Ca2+ regulation in the absence of the iplA geneproduct in Dictyostelium discoideum.  BMC Cell Biol 2005,6(1):13.32. Lusche DF, Malchow D: Developmental control of cAMP-induced Ca2+-influx by cGMP: influx is delayed and reducedin a cGMP-phosphodiesterase D deficient mutant of Dictyos-telium discoideum.  Cell Calcium 2005, 37(1):57-67.33. Van Haastert PJ, Devreotes PN: Chemotaxis: signalling the wayforward.  Nature Reviews Molecular Cell Biology 2004, 5:626-634.34. Bumann J, Wurster B, Malchow D: Attractant-induced changesand oscillations of the extracellular Ca++ concentration insuspensions of differentiating Dictyostelium cells.  J Cell Biol1984, 98:173-178.35. Veltman DM, de Boer JS, van Haastert PJM: Chemoattractant-stimulated calcium influx in Dictyostelium discoideum doesnot depend on cGMP.  Biochim Biophys Acta 2003, 1623:129-134.36. Hall AL, Schlein A, Condeelis J: Relationship of pseudopod exten-sion to chemotactic hormone-induced actin polymerizationin amoeboid cells.  J Cell Biochem 1988, 37:285-299.37. Zarrinpar A, Bhattacharyya RP, Lim WA: The structure and func-tion of proline recognition domains.  Science's Stke [ElectronicResource]: Signal Transduction Knowledge Environment 2003,2003(179):RE8.38. Insall R, Kuspa A, Lilly PJ, Shaulsky G, Levin LR, Loomis WF, Devre-otes P: CRAC, a cytosolic protein containing a pleckstrinhomology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.  JCell Biol 1994, 126:1537-1545.39. Franca-Koh J, Devreotes PN: Moving forward: mechanisms ofchemoattractant gradient sensing.  Physiology 2004, 19:300-308.40. Pak Y, Pham N, Rotin D: Direct binding of the beta1 adrenergicreceptor to the cyclic AMP-dependent guanine nucleotideexchange factor CNrasGEF leads to Ras activation.  MolecularAdditional File 3Chemotaxis of HSB61 starved and pulsed with cAMP for 10 hours.mov: 9.9 MBClick here for file[http://www.biomedcentral.com/content/supplementary/1471-2121-6-43-S3.mov]Page 17 of 18(page number not for citation purposes)Biology 2000, 7(12):1178-1184.& Cellular Biology 2002, 22(22):7942-7952.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Cell Biology 2005, 6:43 http://www.biomedcentral.com/1471-2121/6/4341. van Haastert PJM, van der Heijden PR: Excitation adaptation anddeadaptation of the cAMP mediated cGMP response in Dic-tyostelium discoideum.  J Cell Biol 1983, 96:347-353.42. Uchida KSK, Kitanishi-Yumura T, Yumura S: Myosin II contributesto the posterior contraction and the anterior extension dur-ing the retraction phase in migrating Dictyostelium cells.  JCell Sci 2003, 116:51-60.43. Moores SL, Sabry JH, Spudich JA: Myosin dynamics in live Dicty-ostelium cells.  Proc Natl Acad Sci USA 1996, 93:443-446.44. Stites J, Wessels D, Uhl A, Egelhoff T, Shutt D, Soll DR: Phosphor-ylation of the Dictyostelium myosin II heavy chain is neces-sary for maintaining cellular polarity and suppressing turningduring chemotaxis.  Cell Motil Cytoskel 1998, 39:31-51.45. Postma M, Bosgraaf L, Loovers HM, van Haastert PJM: Chemotaxis:signalling modules join hands at front and tail.  EMBO Rep 2004,5:35-40.46. Bosgraaf L, Russcher H, Snippe H, Bader S, Wind J, van Haastert PJM:Identification and characterization of two unusual cGMP-stimulated phosphodiesterases in Dictyostelium.  Mol Biol Cell2002, 13:3878-3889.47. Steimle PA, Licate L, Cote GP, Egelhoff TT: Lamellipodial localiza-tion of Dictyostelium myosin heavy chain kinase A is medi-ated via F-actin binding by the coiled-coil domain.  FEBS Lett2002, 516:58-62.48. Roelofs J, van Haastert PJM: Characterization of two unusualguanylyl cyclases from Dictyostelium.  J Biol Chem 2002,277:9167-9174.49. Wu LJ, Valkema R, van Haastert PJM, Devreotes PN: The G proteinbeta subunit is essential for multiple responses to chemoat-tractants in Dictyostelium.  J Cell Biol 1995, 129:1667-1675.50. Roelofs J, Loovers HM, van Haastert PJM: GTPgammaS regula-tion of a 12-transmembrane guanylyl cyclase is retainedafter mutation to an adenylyl cyclase.  J Biol Chem 2001,276:40740-40745.51. Watts DJ, Ashworth JM: Growth of myxameobae of the cellularslime mould Dictyostelium discoideum in axenic culture.  Bio-chemical Journal 1970, 119(2):171-174.52. Bozzaro S, Merkl R, Gerisch G: Cell adhesion: its quantification,assay of the molecules involved, and selection of defectivemutants in Dictyostelium and Polysphondylium.  Meth Cell Biol1987, 28:359-385.53. Devreotes P, Fontana D, Klein P, Sherring J, Theibert A: Transmem-brane signaling in Dictyostelium.  Methods in Cell Biology 1987,28:299-331.54. Bozzaro S, Roseman S: Adhesion of Dictyostelium discoideumcells to carbohydrates immobilized in polyacrylamide gels.II. Effect of D-glucoside derivatives on development.  J BiolChem 1983, 258:13890-13897.55. Peracino B, Borleis J, Jin T, Westphal M, Schwartz JM, Wu LJ, BraccoE, Gerisch G, Devreotes P, Bozzaro S: G protein beta subunit-nullmutants are impaired in phagocytosis and chemotaxis dueto inappropriate regulation of the actin cytoskeleton.  J CellBiol 1998, 141:1529-1537.56. Schaloske R, Sordano C, Bozzaro S, Malchow D: Stimulation of cal-cium influx by platelet activating factor in Dictyostelium.  JCell Sci 1995, 108:1597-1603.57. Gerisch G, Hess B: Cyclic-AMP-controlled oscillations in sus-pended Dictyostelium cells: Their relation to morphoge-netic cell interactions.  Proc Natl Acad Sci USA 1974, 71:2118-2122.58. Lilly PJ, Devreotes PN: Identification of CRAC, a cytosolic reg-ulator required for guanine nucleotide stimulation of adeny-lyl cyclase in Dictyostelium.  Journal of Biological Chemistry 1994,269(19):14123-14129.59. Van Haastert PJM: The modulation of cell surface cAMP recep-tors from Dictyostelium discoideum by ammonium sul-phate.  Biochim Biophys Acta 1985, 845(2):254-260.60. Sutoh K: A transformation vector for dictyostelium discoi-deum with a new selectable marker bsr.  Plasmid 1993,30(2):150-154.61. Pang KM, Lynes MA, Knecht DA: Variables controlling theexpression level of exogenous genes in Dictyostelium.  Plasmid1999, 41:187-197.62. Nellen W, Datta S, Reymond C, Sivertsen A, Mann S, Crowley T, Fir-tel RA: Molecular biology in Dictyostelium: tools and applica-63. Southern EM: Detection of specific sequences among DNAfragments separated by gel electrophoresis.  Journal of Molecu-lar Biology 1975, 98(3):503-517.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 18 of 18(page number not for citation purposes)tions.  Meth Cell Biol 1987, 28:67-100.


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items