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RasG signaling is important for optimal folate chemotaxis in Dictyostelium Chattwood, Alex; Bolourani, Parvin; Weeks, Gerald Apr 17, 2014

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RESEARCH ARTICLE Open AccessRasG signaling is important for optimal folatechemotaxis in DictyosteliumAlex Chattwood*, Parvin Bolourani and Gerald WeeksAbstractBackground: Signaling pathways linking receptor activation to actin reorganization and pseudopod dynamicsduring chemotaxis are arranged in complex networks. Dictyostelium discoideum has proven to be an excellentmodel system for studying these networks and a body of evidence has indicated that RasG and RasC, members ofthe Ras GTPase subfamily function as key chemotaxis regulators. However, recent evidence has been presentedindicating that Ras signaling is not important for Dictyostelium chemotaxis. In this study, we have reexamined therole of Ras proteins in folate chemotaxis and then, having re-established the importance of Ras for this process,identified the parts of the RasG protein molecule that are involved.Results: A direct comparison of folate chemotaxis methodologies revealed that rasG-C- cells grown in associationwith a bacterial food source were capable of positive chemotaxis, only when their initial position was comparativelyclose to the folate source. In contrast, cells grown in axenic medium orientate randomly regardless of their distanceto the micropipette. Folate chemotaxis is restored in rasG-C- cells by exogenous expression of protein chimerascontaining either N- or C- terminal halves of the RasG protein.Conclusions: Conflicting data regarding the importance of Ras to Dictyostelium chemotaxis were the result ofdiffering experimental methodologies. Both axenic and bacterially grown cells require RasG for optimal folatechemotaxis, particularly in weak gradients. In strong gradients, the requirement for RasG is relaxed, but only inbacterially grown cells. Both N- and C- terminal portions of the RasG protein are important for folate chemotaxis,suggesting that there are functionally important amino acids outside the well established switch I and switch IIinteraction surfaces.Keywords: Ras GTPase, Folate chemotaxis, SignallingBackgroundDirectional cell movement is important throughout thelife cycle of multicellular organisms, from axon guidanceduring embryogenesis to wound healing in adults [1,2].Understanding the mechanisms by which cells move willultimately require a detailed knowledge of the compo-nents that regulate the behaviour of force-generatingcytoskeletal proteins, such as actin and myosin.The life cycle of the social amoeba, Dictyosteliumdiscoideum, is dependent on directional cell movement.In the growth phase, amoebae are attracted to folatereleased from their bacterial prey [3]. When starved,amoebae establish a signal relay system based on cAMPthat causes them to aggregate together into a multicel-lular structure. In both cases, two Ras proteins, RasGand RasC, have been shown to play central roles [4-7].The localization of activated Ras to the front of the cellis one of the first responses to chemoattractant stimula-tion [8]. A recent study has shown that activation firstoccurs across the entire plasma membrane but progres-sively becomes more confined to the leading edge [9].Furthermore, results from a detailed analysis of Rasactivation in various rasGEF and rasGAP mutants sup-port the idea that multiple Ras isoforms, such as RasGand RasC drive chemotaxis [9-11].Vegetative cells lacking RasG exhibit a complex pheno-type; reduced growth, reduced macropinocytosis, defectsin cytokinesis, a disorganized cytoskeleton, reduced motil-ity and reduced folate chemotaxis [5,12,13]. Removal ofRasC likewise results in reduced folate chemotaxis, though* Correspondence: alexchat@mail.ubc.caDepartment of Microbiology and Immunology, University of BritishColumbia, 1365, Life Sciences Centre 2350, Health Sciences Mall, V6T 1Z3Vancouver, BC, Canada© 2014 Chattwood et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative 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 credited. The Creative Commons PublicDomain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in thisarticle, unless otherwise stated.Chattwood et al. BMC Cell Biology 2014, 15:13http://www.biomedcentral.com/1471-2121/15/13the effect is somewhat milder compared to the lossof RasG [6]. However, when both RasG and RasC areremoved in combination, the mutant cells exhibit identicalphenotypes to those of rasG- cells, except that they aretotally incapable of directional movement [7,14]. Thissuggests that RasG and RasC synergise to regulate folatechemotaxis but that RasG alone regulates cytoskeletalorganization and motility [7]. Downstream of Ras acti-vation, three pathways have been identified that areactivated with similar kinetics; PI3K, TORC2 and sGC[15]. Furthermore, cells lacking RasG and/or RasC exhibitreduced activation of PI3K and TORC2 pathways, pre-sumably because effective signaling requires direct inter-action between these proteins and active Ras [8,11,16,17].Surprisingly, however, none of these pathways are essentialfor chemotaxis to folate and instead serve only accessoryroles in signal amplification [7]. In fact, recent evidencesuggests that PI3K signaling is required only for macropi-nocytosis, and can actively inhibit folate chemotaxis[18,19]. A more likely candidate connecting Ras activationto the chemotaxis apparatus is PiKI, which produces PIP2.Cells lacking this protein display normal Ras activationbut fail to initiate Ras-dependent responses or orientatein cAMP gradients [20]. Thus, while the genetic analysisof mutants is complicated by apparent redundancybetween isoforms and an incomplete knowledge of thebranching downstream pathways, there is a generalconsensus that Ras plays a significant role in signalingduring directional sensing and migration [21]. This con-sensus view has, however, been challenged by a recentstudy that has shown rasG-/rasC- mutant cells are fullycapable of folate chemotaxis, although different experi-mental conditions were used [22].One consequence of removing RasG alone or bothRasG and RasC from cells is the up-regulation of RasD[5,13], a protein whose expression is normally restrictedto the developmental stages of the Dictyostelium lifecycle [23]. RasD and RasG share 83% identity and differby only 3 amino acids in the N-terminal 106 residues ofthe protein, with no variation in effector switch I orswitch II domains. It is clear that this up-regulation isinsufficient to prevent the observed defective vegetativecell phenotypes of rasG- and rasG-/rasC- mutant cells.However, the addition of exogenous RasD expressionrescues the growth and cytokinesis defects, but not themotility and folate chemotaxis defects of these cells [5,13].In this study we have directly compared folate chemo-taxis in rasG-/rasC- cells, using the original experimen-tal conditions [5,7] and the conditions used in the morerecent study [22]. We have confirmed that RasG isimportant for optimal folate chemotaxis, and have thenexplored, using protein chimeras of RasG and RasD,which portions of the RasG molecule contribute tofolate chemotaxis.Results and discussionRequirement of Ras signaling in folate chemotaxis invegetative cells depends upon growth conditionsThe recent observation indicating the absence of a rolefor Ras proteins in folate chemotaxis [22] is at odds withearlier observations that a rasG-/rasC- double knockoutstrain displayed zero chemotaxis towards a folate-filledmicropipette [5,7]. In order to try to reconcile these con-flicting results, we tested whether methodological differ-ences between the two studies affect the chemotacticaccuracy of the rasG-/rasC- cells. One important differ-ence was that the earlier studies used cells grown axen-ically (the term “axenic” is used to refer to cells thatobtain nutrients from liquid medium, in the absence ofbacteria), while the more recent study used cells grownon bacteria. Therefore, we directly compared the folatechemotaxis indices of cells cultured axenically and onbacteria.Our results showed that the mean chemotaxis index ofrasG-/rasC- cells was significantly increased by the shiftfrom axenic to bacterial growth (Figure 1, mean diff. =0.227, 95% CI [0.092, 0.362], p = <0.0001). This result isconsistent with the recent conclusion that rasG-/rasC-cells are capable of positive chemotaxis [22]. We noted,however, that there was still a significant reduction infolate chemotaxis between JH10 cells and rasG−/rasC-cells grown on bacteria (Figure 1, mean diff. = −0.183,95% CI [−0.066, −0.3], p = <0.001). Furthermore, therewas negligible chemotaxis by rasG-/rasC- cells relativeto control JH10 cells, when cells were grown axenically(Figure 1, mean diff. = −0.336, 95% CI [−0.219, −0.452],p = <0.00001), supporting the earlier conclusions [5,7].Figure 1 The effect of axenic vs. bacterial growth on folatechemotaxis. Light grey circles show the average chemotaxis indexof individual control JH10 [n = 80] and rasG-/rasC- cells [n = 40],grown either in axenic medium or on bacteria. Error bars indicatethe 95% confidence interval of the mean (middle black bar).Chattwood et al. BMC Cell Biology 2014, 15:13 Page 2 of 7http://www.biomedcentral.com/1471-2121/15/13Our data, therefore, indicate that Ras signaling is im-portant for optimal chemotaxis to folate regardless ofgrowth conditions, but that this requirement is dimin-ished by growth on bacteria.Redundancy amongst Ras proteins does not explain theenhanced folate chemotaxis of bacterially grown rasG-/rasC- cellsOne possible explanation for the enhanced chemotaxisphenotype of rasG-/rasC- cells is that growth on bacteriainduces cellular changes that render RasG and RasC lessimportant during folate chemotaxis compared to other Rasproteins. The Dictyostelium genome encodes 11 ras genes.Of these, 5 (RasG, RasC, RasD, RasB, RasS) have been par-tially characterised, and all (except RasS) have been de-tected by specific antibodies during the growth phase andshown to have different mobilities on electrophoresis gels[13,24]. Extremely low transcript levels argue that theremaining 6 ras gene products are unlikely to be detectableby Western Blot (DictyExpress). Therefore, the levels andidentities of different Ras proteins can be identified using anon-specific Pan-Ras antibody.Firstly, there was no evidence of any novel Ras isoformin the bacterially grown rasG-/rasC- cells when com-pared to their axenic counterparts (Figure 2). This resultsuggests that any Ras-mediated improvements to folatechemotaxis would manifest as changes in the expressionlevel of the Ras proteins already present in these cells.It has been shown previously that RasD expression isconsiderably enhanced when the rasG gene is deleted[5,13], and this enhanced expression is also detected bythe Pan-Ras antibody. RasD levels were undetectablein the JH10 cells but clearly present in both rasG- andrasG-/rasC- cells grown axenically and on bacteria(Figure 2). In addition, bacterial growth of these cells wascorrelated with qualitative increases in RasD levels.This increase cannot explain the improvement in folatechemotaxis of rasG−/rasC− cells observed by a switchfrom axenic to bacterial growth, since exogenous RasDexpression does not rescue the chemotactic defects ofrasG−/rasC− cells [5,13].Surprisingly, there was a comparative decrease in RasGlevels in JH10 cells grown on bacteria. This clearly has noimpact on folate chemotaxis, since their folate chemotac-tic indexes are not significantly different (Figure 1), andmay instead be related to the significant differences ingrowth rates between the bacterially and axenically growncells.Finally, it is also unlikely that there are increased levelsof active Ras in bacterially grown rasG−/rasC− cells,because membrane recruitment of RBD-GFP remainslow in cells stimulated with folate [22]. Thus, there isno evidence that the improved chemotactic accuracy ofbacterially grown rasG−/rasC− cells involves novel Rassignaling pathways or the modulation of currently exist-ing ones.Initial distance from micropipette affects folatechemotaxis of bacterially grown rasG-/rasC- cellsThere is an additional difference in the folate chemotaxismeasurements between the earlier and more recentstudies. In the more recent study [22], folate chemotaxiswas measured in a field of cells that appeared to be atconsiderably higher cell density than was used for theearlier measurements [5,7] and consequently the chemo-tactic index was measured only for cells close to the tip.Chemoattractant gradients decrease exponentially at in-creasing distances from the tip such that cells close tothe tip experience high concentrations and steep gradi-ents of folate, whereas those further away experiencelower concentrations and shallower gradients [25]. Thishas been shown to influence chemotactic measurements[7]. We therefore controlled for the cell density of therasG-/rasC- cells and looked at the effect of distancefrom the micropipette at T0 on the determination of thefolate chemotaxis index. As shown in Figure 3, individ-ual rasG-/rasC- cells grown on bacteria displayed posi-tive chemotaxis below an initial distance of 200 μm(average chemotactic index of 0.33), but negligible levelsof chemotaxis (average chemotactic index of 0.08) above adistance of 200 μm. Over the same distance range, JH10cells grown on bacteria exhibited no variation (Figure 3).Thus, initial distance of the cell from the micropipette isan important factor determining whether or not bacter-ially grown rasG−/rasC− cells will exhibit directionalmovement. In contrast, rasG−/rasC− cells grown axen-ically displayed negligible chemotaxis at all distancesfrom the micropipette (Figure 3), confirming previouslypublished data [7]. This result reinforces the idea thatRas signaling is crucial for folate chemotaxis of axenic-ally grown cells. Furthermore, we show that Ras signal-ing is not absolutely essential for the chemotaxis ofbacterially grown cells, but that its role becomes in-creasingly important at either lower concentrations orshallower gradients of folate.Figure 2 Total Ras expression in axenic and bacterially growncells. Levels determined with Pan-Ras antibody. The lower Mr bandsin the rasG- and rasG-rasC- lanes represents up-regulated RasD (arrow).Chattwood et al. BMC Cell Biology 2014, 15:13 Page 3 of 7http://www.biomedcentral.com/1471-2121/15/13The above results indicate that there is a clear differ-ence between the requirements of Ras for folate chemo-taxis in axenically and bacterially grown cells. Therehave been several reports detailing morphological, meta-bolic and transcriptional differences between axenicallyand bacterially grown cells [26,27]. There are also effectson cell motility. First, bacterially grown cells move fasterthan axenic cells in random motility assays [28-30]. In-deed, our own measurements show that bacteriallygrown JH10 and rasG-/rasC- cells migrate at higher vel-ocities than their axenically grown counterparts duringfolate chemotaxis (Additional file 1: Figure S1). Second,a recent study has shown that transfer of the back-ground strain AX2 from axenic to bacterial growth im-proves chemotaxis in linear folate gradients [19]. In thesame paper, PIP3-dependent macropinocytosis, the pre-dominant mode of feeding in amoebae grown axenic-ally, was identified as an inhibitor of chemotaxis. Wedid not observe a significant difference between thechemotactic indices of JH10 cells grown in axenicmedium or on bacteria (Figure 1), possibly owing tostrain-specific differences. However, there was a cleardifference between folate chemotaxis of axenic andbacterially grown rasG-/rasC- cells (Figure 1). Whilstaxenically grown rasG−/rasC− cells exhibit reduced macro-pinocytosis (unpublished observations), there may still besufficient activity to severely inhibit folate chemotaxis.Although the effect of macropinocytosis inhibitionmay explain some of our observations, it does not ex-plain why rasG-/rasC- cells exhibit reduced chemotaxisregardless of their growth condition. One possibility isthat axenically grown cells are relatively unpolarized,and only become polarized when exposed to folate gra-dients (R. Insall, personal communication). Ras signal-ing is important for initiating and reinforcing decisionsto polarize the cytoskeleton [9] and axenically grownrasG-/rasC- cells may be less capable of amplifying sig-nals sufficiently to be able to orient towards the chemo-tactic signal. In contrast, Ras-dependent amplificationof the chemotactic signal in a strong folate gradient isimportant for optimal chemotaxis of bacterially grownrasG-/rasC- cells, but not essential. Interestingly, Rassignaling is still of prime importance for the bacteriallygrown rasG-/rasC- cells in shallow folate gradients.Specificity of the RasG requirement for optimumchemotaxisWe showed previously that exogenous expression ofRasD was incapable of restoring folate chemotaxis inrasG-/rasC- cells [5,13]. To understand what makesRasG uniquely required for chemotaxis, we decided togenerate chimeras of RasG and RasD that would assessthe relative importance of the 3 amino acids in theN-terminal portion of the RasG protein and the differ-ences in residues in the C-terminal half of the protein. Forthis, a PCR-based approach was used in which primerswere designed to generate a product that would leave anoverhanging end containing sequence to which a corre-sponding product could ligate. The product, designatedRasD1G2, corresponds to the N-terminal 104 residuesof RasD ligated to the C-terminal 86 residues of RasG.The product, designated RasG1D2, corresponds to theN-terminal 104 residues of RasG ligated to the C-terminal84 residues of RasD (Note: the total sequence lengths arenot the same because RasG contains two additional aminoacids). Each of these products was cloned into an exogen-ous expression vector downstream of the rasG promoterand transformed into rasG-/rasC- cells. Intact RasD andRasG were also transformed so that there was an experi-mental baseline to which our chimera data could be com-pared. G418-resistant clones were isolated and subjectedto Western blotting with specific RasD or RasG antibodiesto confirm that all the proteins were expressed at similarlevels (Figure 4A).As shown previously, expression of RasG in rasG-/rasC-cells almost fully rescued the folate chemotaxis defect,while RasD expression had a minimal effect on chemo-taxis (Figure 4B). The expression of either the RasD1G2or RasG1D2 constructs in rasG-/rasC- cells significantlyimproves folate chemotaxis (Figure 4B). However, a statis-tical comparison between JH10 cells and RasD1G2 andRasG1D2 expressing cells reveals that chemotaxis is fullyrestored only by RasG1D2 (Figure 4B, mean diff. = 0.023,95% CI [−0.105, 0.151], p = >0.05), and not RasD1G2 pro-teins (Figure 4B, mean diff. = 0.155, 95% CI [−0.027,0.283], p = <0.05). This result suggests 1) that both the 3altered amino acids in the N-terminal portion and theentire C-terminal portion make important contributionsto RasG-mediated chemotaxis and 2) the N-terminal0 200 400 600-0.50.00.51.0Chemotactic IndexDistance from tip at T0 (μm)Figure 3 The effect of distance on folate chemotaxis ofbacterially grown rasG-/rasC- cells. Chemotaxis index values plottedagainst distance from micropipette at T0. N = 40: Bacterially grown JH10cells (open grey circles); bacterially grown rasG-rasC- cells (closed blackcircles); axenically grown rasG-rasC- cells (closed grey circles).Chattwood et al. BMC Cell Biology 2014, 15:13 Page 4 of 7http://www.biomedcentral.com/1471-2121/15/13portion is perhaps more important for this functionthan the C-terminal portion. Likewise, an earlier studyfound that both the N-terminal and C-terminal portionsof the RasC molecule were important for adenylatecyclase activation during the aggregation of starving cellstowards the chemoattractant, cAMP [31]. It was suggestedthat both the interaction of RasC with a specific down-stream effector through the N-terminal portion of themolecule and the subcellular membrane localizationthrough its C-terminal portion were important for func-tion. Similar conclusions may be reached regarding thespecificity of RasG for folate chemotaxis in vegetativecells. However, it is important to note that the differ-ences between N-terminal portions of RasG and RasDnumber only 3 amino acids. Moreover, each of thesedifferences is conservative (Ser > Thr, Asp > Glu, Tyr >Phe), and located at positions outside of the normalswitch I and switch II interaction surfaces.Understanding the functional specificity of differentRas isoforms is a hurdle that must be overcome to developefficacious inhibitors of Ras signaling [32,33]. Proteinchimeras composed of subfamily isoforms can be used toexplore portions of protein molecules that are importantfor specific functions. Dictyostelium RasD and RasG arehighly homologous to human Ras proteins (64% and 67%compared to H-Ras; 65% and 70% compared to K-Ras).Therefore, an insight into what makes RasG and RasDfunctionally distinct from each other may open up newavenues of inquiry in higher organisms.ConclusionRasG is required for optimal chemotaxis, regardless ofgrowth condition. Increased initial distance from thechemoattractant source is correlated with reducedchemotactic accuracy, suggesting that RasG is particularlyimportant for directional cell migration in weak gradients.In strong gradients, the requirement for RasG is relaxed,but only in bacterially grown cells. The role of RasG infolate chemotaxis is unique, and cannot be replaced by the83% identical, RasD molecule. Both N- and C- terminalportions of the RasG protein contribute to folate chemo-taxis, suggesting that there are functionally importantamino acids outside the well established switch I andswitch II interaction surfaces.MethodsCell culture and maintenanceFor axenic growth, all strains were cultured in HL-5medium (per litre: 15.4 g glucose, 14.3 g bactopeptone,7.15 g yeast extract, 0.96 g Na2HPO4, 0.49 g KH2PO4)containing 50 mg/ml streptomycin (Sigma, USA) and50 mg/ml ampicilin (Sigma, USA). Strain JH10 was furthersupplemented with 100 μg/ml thymidine (Sigma, USA).Strains transformed by electroporation (www.dictybase.org) with 20 μg exogenous plasmid were selected andmaintained in HL-5 with 10 μg/ml G418 (Invitrogen,Carlsbad CA). For bacterial growth, clearing plateswere prepared. In which, 5e5 amoebae were plated inassociation with 400 μl of a thick suspension of Klebsiellaaerogenes and kept in a dark, moist box at 22°C. Cells weredeemed ready for experimentation once the amoebae hadeaten the majority of bacteria, indicated by a change ofsurface texture in the petri dish; from opaque and matteto transparent and glassy. Preparation of cells grownunder different conditions for experimentation was as fol-lows: Axenic cells were washed from sub-confluent tissuecultures plates (Nunc, Rochester NY) containing HL-5.Bacterially grown cells were harvested into HL-5 fromclearing plates and the remaining bacteria removed by 3×5 min centrifugation steps at 1000 rpm. Axenic and bac-terially grown amoebae were henceforth treated identicallyin all subsequent experiments.ABFigure 4 Both N- and C- terminal halves of RasG are requiredfor optimal folate chemotaxis of axenically grown cells.(A) Expression of RasG/RasD protein chimeras detected by bindingof RasG and RasD antibodies specific to the C-terminus. (B) Chemotaxisindex protein chimeras and controls [n = 40]. Error bars indicatethe 95% confidence interval of the mean. Statistical significance:ns = P > 0.05; *** = P < 0.001; **** = P < 0.0001. Light grey circlesshow average chemotaxis index of each cell. All strains were grown inaxenic conditions.Chattwood et al. BMC Cell Biology 2014, 15:13 Page 5 of 7http://www.biomedcentral.com/1471-2121/15/13Chimera constructsAll Ras proteins were cloned into vector #188, a modifiedversion of pBS KS (Promega, WI) in which the neomycinresistance cassette and a genomic fragment containing theRasG promoter and coding sequence have been inserted.Genomic RasG was replaced with coding sequence usingBglII and XhoI restriction sites, such that the RasG pro-moter drove the expression of the cloned fragment. RasDand RasG constructs were generated by PCR from pGEM-T-Easy (Promega, WI) templates containing RasD andRasG cDNA. These vectors also served as templates togenerate the PCR fragments that were subsequentlyligated to form RasD1G2 and RasG1D2 constructs, usingthe method and thermocycling parameters detailed in[31]. Primers used: D1-F_BglII (5′-CGCAGATCTATGACAGAATATAAATTA-3′), D2-F (5′-AAAGATAGAGTACCATTGATTTTGG-3′), D2ovr-G1R (5′-CAATGGTACTCTATCTTTATCCTTAACTCTAAGAATTTGTTC-3′),D2-R_XhoI (5′-AGGCTCGAGTTATAAAATTAAACATTG-3′), G2-F (5′-AAGGATAGAGTACCAATGATTGTCG-3′), G2-R_XhoI (5′-CGTCTCGAGTTATAAAAGAGTACAAG-3′), G2ovr_D1R (5′-CATTGGTACTCTATCCTTGTCTTTAACTCTTAGAATTTGTTC-3′). Note thatitalics designate restriction sites and underlined sym-bolizes overhangs. Primer combinations: RasD = D1-F_BglII + D2-R_XhoI; RasG = G1-F_BglII + G2-R_XhoI;RasD1 = D1-F_BglII + G2ovr_D1R; RasD2 = D2-F + D2-R_XhoI; RasG1 =G1-F_BglII + D2ovr_G1R; RasG2 =G2-F +G2-R_BglII. Products D1 +G2 were ligated to generateRasD1G2 and products G1 +D2 were ligated to generateRasG1D2 fragments.Western blotsCells harvested from axenic medium and bacterial clearingplates were resuspended to a density of 1e7 cells/ml in 1×HK-LB (10% glycerol, 150 mM NaCl, 10 mM Na2PO4pH7.2, 10 mM MgCl2, 5 mM NaF, 1 mM Na3VO4, 1 mMEDTA, 1% Triton X-100, 0.05% SDS). Protein concentra-tion was determined by DC Assay (BioRad, CA), 6× SDS-PAGE buffer (350 mM Tris-Cl pH6.8, 30% glycerol, 10%SDS, 0.01% bromophenol blue) was added and sampleswere boiled for 5mins. 20 μg protein were loaded intoeach lane and fractionated by SDS-PAGE. After electro-phoresis, proteins were transferred to a Hybond-Pmembrane (Amersham) and equal loading was verifiedby Ponceau S (BioRad, CA) staining. Membranes wereblocked with 5% non-fat milk solution, and probed withantibody. To compare Ras expression in axenic andbacterially grown cells, membranes were probed over-night at 4°C with 1:1000 Anti Pan-Ras primary anti-body (CalBiochem cat# op400), followed by 1 hr roomtemperature binding of 1:5000 Anti mouse secondaryantibody (GE Healthcare cat# NA931). To examine theexpression of chimeric Ras proteins, specific RasD andRasG antibodies were used at concentrations of 1:300and 1:500, respectively.Folate chemotaxisCells harvested from axenic medium and bacterial clearingplates were suspended in antibiotic-free HL-5, depositedonto 6 cm tissue culture plates at a density of 4e5 cells/cm2 and allowed to attach for 15 mins. Media was re-placed with 20% HL-5 and an Eppendorf Femtotip micro-pipette filled with 25 mM folate was positioned in thesame focal plane as the cells. Cell movement was capturedat 30s intervals by time-lapse microscopy.Chemotaxis analysisCell tracking was performed on randomly chosen cellsusing the mTrackJ plugin [34] in ImageJ. Coordinate in-formation from each cell was transformed into chemotac-tic metrics in Microsoft Excel and graphed in GraphPadPrism. A single chemotactic index datapoint is the cosineof the angle between a line connecting a cell to the tip attime, n, and a line connecting a cells position at time, n, toits position at n + 1. The mean chemotactic index wasdetermined from the sum total of indices in each track. Atrack was deemed complete if the cell remained in closeproximity to the pipette for four consecutive frames. Ascore of 1 indicates perfect chemotaxis.Additional fileAdditional file 1: Figure S1. The effect of axenic vs. bacterial growthon cell velocity. Error bars indicate the 95% confidence interval of the mean.Light grey circles show average velocity of each individual cell [n = 40].Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAC conceived and designed experiments, acquired data, analyzed andinterpreted data, drafted the manuscript. PB conceived and designedexperiments, acquired data, analyzed and interpreted data, drafted themanuscript. GW conceived and designed experiments, interpreted data,drafted the manuscript. All authors read and approved the final manuscript.AcknowledgementsThis work was funded by a CIHR operating grant awarded to GW.Received: 28 January 2014 Accepted: 14 April 2014Published: 17 April 2014References1. Song HJ, Poo MM: Signal transduction underlying growth cone guidanceby diffusible factors. Curr Opin Neurobiol 1999, 9(3):355–363.2. 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Meijering E, Dzyubachyk O, Smal I: Methods for cell and particle tracking.Methods Enzymol 2012, 504:183–200.doi:10.1186/1471-2121-15-13Cite this article as: Chattwood et al.: RasG signaling is important foroptimal folate chemotaxis in Dictyostelium. BMC Cell Biology 2014 15:13.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitChattwood et al. BMC Cell Biology 2014, 15:13 Page 7 of 7http://www.biomedcentral.com/1471-2121/15/13

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