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BMC Microbiology. 2009 Jan 14;9(1):9
Kemmer et al.
Kemmer, Danielle
McHardy, Lianne M.
Hoon, Shawn
Rebérioux, Delphine
Giaever, Guri
Nislow, Corey
Roskelley, Cal
Roberge, Michel
Background: Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches - analysis of the sensitivity of ρ 0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.
ralssBioMed CentBMC MicrobiologyOpen AcceResearch articleCombining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesisDanielle Kemmer1, Lianne M McHardy1, Shawn Hoon2, Delphine Rebérioux1, Guri Giaever3,4,5, Corey Nislow4,5,6, Calvin D Roskelley7 and Michel Roberge*1Address: 1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada, 2Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA, 3Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, 4Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada, 5Department of Molecular Genetics, University of Toronto, Toronto, Canada, 6Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada and 7Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, CanadaEmail: Danielle Kemmer - dakemmer@interchange.ubc.ca; Lianne M McHardy - liannemarie@gmail.com; Shawn Hoon - shawnh@stanford.edu; Delphine Rebérioux - delphine.reberioux@pierre-fabre.com; Guri Giaever - guri.giaever@utoronto.ca; Corey Nislow - corey.nislow@utoronto.ca; Calvin D Roskelley - roskelly@interchange.ubc.ca; Michel Roberge* - michelr@interchange.ubc.ca* Corresponding author AbstractBackground: Single genome-wide screens for the effect of altered gene dosage on drug sensitivityin the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism ofaction of a drug.Results: Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we showthat a more complete picture of drug action can be obtained by combining different chemicalgenomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast bythis chemical requires a functional mitochondrial electron-transport chain and cytochrome c hemelyase function. However, we find that it does not require genes associated with programmed celldeath in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking andinterferes with vacuolar acidification in yeast and in human cancer cells. These effects can all beascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C.Conclusion: Despite their similar conceptual basis, namely altering drug sensitivity by modifyinggene dosage, each of the screening approaches provided a distinct set of information that, whenintegrated, revealed a more complete picture of the mechanism of action of a drug on cells.BackgroundKnowledge of the different proteins and cellular processesbecause unbiased techniques to sample all possible targetproteins and pathways are currently lacking. The observa-Published: 14 January 2009BMC Microbiology 2009, 9:9 doi:10.1186/1471-2180-9-9Received: 17 July 2008Accepted: 14 January 2009This article is available from: http://www.biomedcentral.com/1471-2180/9/9© 2009 Kemmer 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 17(page number not for citation purposes)affected by chemicals is necessary to rationally guide drugdiscovery and development. This is a difficult challengetion that modifying the amount or activity of a gene prod-uct via mutation, overexpression, downregulation orBMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9deletion can change the response of a cell to a chemical[1,2] raises hope that systematic genome-wide screens ofdrug sensitivity can help uncover direct and indirect drugtargets as well as modifiers of cellular responses to chem-icals.The yeast Saccharomyces cerevisiae is currently the eukaryo-tic model organism with the most comprehensivegenome-wide collections of mutant strains available tomonitor drug sensitivity. S. cerevisiae exists as a haploid oras a diploid. Deleting 1 of the 2 copies of a gene in diploidstrains can reduce its expression, and a set of ~6,000 het-erozygous diploid strains covering nearly all essential andnonessential genes is available. Complete deletion ofnonessential genes eliminates their expression and sets of~4,900 haploid and homozygous diploid deletionmutants are also available. S. cerevisiae can be easily trans-formed and increased gene expression can be achieved byintroducing plasmids containing genomic DNA frag-ments or gene-coding regions controlled by induciblepromoters [3]. The unicellular nature of yeast and its abil-ity to grow on liquid or solid media also make it amenableto high-throughput drug studies.A number of studies have shown that reducing the copynumber of essential or nonessential genes from 2 to 1 indiploid cells may increase the sensitivity of the cell to adrug (termed drug-induced haploinsufficiency) and canpoint to candidate target genes [4-6]. Haploid orhomozygous diploid deletion collections contain onlydeletions of nonessential genes. Screening these collec-tions for hypersensitivity to a small molecule revealsgenes that buffer the drug target pathway, not the directdrug targets and comparison of the profile of chemical-genetic synthetic lethality with a compendium of chemi-cal-genetic or genetic interaction profiles can aid in deci-phering its targets [7,8]. Increased gene expression canlead to suppression of drug sensitivity and also reveal tar-get genes [3,9]. Studies of the mechanism of action ofdrugs using genome-wide approaches in yeast havetended to focus on 1 of these 3 approaches [3,5,8]. Whileeach generally reveals important clues, they draw only apartial picture of the mechanism of action of chemicals.For example, a drug-induced haploinsufficiency screen ofthe cancer cell invasion inhibitor dihydromotuporamineC (dhMotC) showed that the compound targets sphingol-ipid biosynthesis and affects the actin cytoskeleton [6],but did not reveal whether other cellular functions wereaffected and gave no indication of cell death mechanismsinvolved.Genome-wide studies of drug mechanism of action havemainly concentrated on nuclear-encoded genes. Genesmitochondrial proteins, have not received as much atten-tion. Yet mitochondria are recognized as important regu-lators of cell death in addition to their central role inenergy production [10]. Although yeast displays onlysome of the characteristics of apoptosis described inhumans, many cellular features of the cell death pathwayin mammalian cells have been identified in yeast [11].Therefore, it may also be important to examine the contri-bution of the mitochondrial genome to drug action.Here we first examine the importance of the mitochon-drial genome to drug sensitivity using ρ0 petite strainsdeleted of mitochondrial DNA. We then examine thevalue to elucidating the mechanism of action of dhMotCof combining screening of ρ0 cells with 3 genome-widescreening approaches: drug-induced haploinsufficiency,chemical-genetic synthetic lethality and suppression ofdrug sensitivity by increased gene expression. We find thatdespite their similar conceptual basis, namely alteringdrug sensitivity by modifying gene dosage, the 3approaches can provide distinct sets of information that,when integrated, reveal a much more complete picture ofthe spectrum of effects of a drug on cells.Results and discussionScreen for mitochondria-dependent inhibitors of yeast growthHalo assays, traditionally used in antibacterial screens,can be used to assess cytotoxic properties of chemicals inyeast [12]. Fungistatic and fungicidal chemicals spottedonto plates containing a lawn of S. cerevisiae growing insoft agar cause zones of growth inhibition (halos) that areeasily detected by visual inspection. Robotic pinning ena-bles high-density arraying of compounds for increasedthroughput.We used the halo assay to screen approximately 3,500FDA-approved drugs and bioactive chemicals [13] as wellas in-house chemicals for inhibition of yeast growth.Chemicals were pin-transferred onto agar containing thewild type yeast strain BY4741 [14] or strain FY1679-28C/TDEC [15] with deletion of 2 transcription factors, PDR1and PDR3, that regulate a wide range of multidrug resist-ance genes, to increase the likelihood of identifying activecompounds. To determine the effect of functional mito-chondria on drug sensitivity, the screen was also carriedout on respiratory-deficient ρ0 petite mutants of the 2strains. The strains lacking functional mitochondria weregenerated by propagating cells in the presence of ethidiumbromide, resulting in the selective loss of the mitochon-drial genome, including several essential components ofthe electron-transport chain, which renders cells respira-tory-deficient [16]. The ρ0 petite strains were unable toPage 2 of 17(page number not for citation purposes)encoded by mitochondrial DNA, which include compo-nents of the mitochondrial translational machinery and 8grow on glycerol, a nonfermentable carbon source, con-BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9firming their inability to generate ATP by mitochondrialoxidative phosphorylation (data not shown).Plates were inspected after 48 h incubation at 30°C andhalos > 2 mm in diameter were scored. 51 chemicalsinhibiting the growth of FY1679-28C/TDEC were identi-fied (Table 1), 39 of which also inhibited the growth ofBY4741. Only 4 chemicals affected the growth of wildtype and ρ0 cells differently. Suloctidil, myriocin, dhMotCand antimycin A inhibited respiratory-competent strainsbut failed to inhibit the growth of the ρ0 strains (Figure 1Aand 1B). Suloctidil, myriocin and dhMotC caused theappearance of clear halos that did not contain observablegrowth, characterizing these compounds as fast-actingantifungals, while antimycin A enabled slow growthwithin the area of the halo (Figure 1B). A single chemical,identified as bithionol, strongly inhibited the growth ofthe ρ0 but inhibited the growth of wild type yeast veryweakly (Figure 1C). Since antimycin A directly blocks themitochondrial electron-transfer chain, a difference in drugresponse between wild type and ρ0 strains was to beexpected and this compound was not studied further.The results of the halo screen were confirmed andextended using a quantitative liquid growth assay. Suloc-tidil (50 μM), myriocin (0.25 μM) and dhMotC (60 μM)all inhibited the growth of the wild type strain while ρ0cells were resistant (Table 2). P0 strains have previouslybeen shown to have increased expression of multidrugresistance genes [17], which could have explained theirincreased resistance to the 4 chemicals. However, this isnot the case since increased resistance was also observedin the ρ0 strain lacking PDR1 and PDR3, that cannotexpress multidrug resistance genes, in agar (Table 1), aswell as in liquid assay (data not shown). Therefore, resist-ance to the growth inhibitory effect of the chemicals is dueto the lack of mitochondrial function.Myriocin binds to and inhibits serine palmitoyltrans-ferase, responsible for the first committed step of de novosynthesis of sphingosine, ceramide and complex sphin-golipids [18]. DhMotC, an inhibitor of tumor cell inva-sion [19], also inhibits sphingolipid biosynthesis andgenes of the sphingolipid biosynthesis pathway showdhMotC-induced haploinsufficiency [6]. Interestingly,suloctidil was recently shown to inhibit acid sphingomy-elinase, a lysosomal enzyme catalyzing the degradation ofsphingolipids and generating ceramide, which can bemetabolised into sphingosine [20].These results show that the majority of chemicals thatinhibit yeast growth do not require functional mitochon-dria to exert their effect but that 3 compounds affectingsphingolipid metabolism all require functional mito-Halo screen of chemical collection with ρ+ and ρ0 FY1679-28C/TDECFigure 1Halo screen of chemical collection with ρ+ and ρ0 FY1679-28C/TDEC. (A) Suloctidil and myriocin selectively inhibit Page 3 of 17(page number not for citation purposes)growth of ρ+ cells; (B) Antimycin A selectively slows growth of ρ+; (C) Increased sensitivity of ρ0 cells to bithionol.BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9chondria to inhibit growth. We then further explored therequirement for functional mitochondria in the mecha-nism of action of 1 of these chemicals, dhMotC, usinggenetic screens and biological assays.Prolonged exposure to dhMotC kills yeastGrowth-inhibitory compounds can reversibly prevent cellproliferation (cytostatic activity) or induce death (cyto-cidal activity). To distinguish between these outcomes,cells were exposed to inhibitory concentrations ofdhMotC in liquid culture for different times and equal cellnumbers were plated onto drug-free agar plates for 2 daysat 30°C. Cells exposed to dhMotC for 1, 3 or 6 hours allformed the same number of colonies as untreated cul-tures. However, exposure to dhMotC for 20 h resulted inno colony growth (Figure 2). These observations showthat dhMotC exposure initially triggers a reversible growtharrest that eventually leads to cell death after longer expo-sure.DhMotC sensitivity suppressor screen reveals genes involved in mitochondrial functionScreens using increased gene dosage, relying on theassumption that increased levels of a protein targeted by adrug increase resistance to the drug, can help identify spe-cific drug targets [9]. Drug sensitivity suppressor screenscan be carried out with pooled genomic library transform-ants, leading to enrichment of resistant strains and deple-tion of hypersensitive strains, relative to untreated pools.Analysis of relative strain sensitivity is performed byhybridization of labelled DNA to an oligonucleotide tagarray [21].A pooled collection of yeast strains expressing genes froma random genomic DNA fragment library was exposed todhMotC and resistant strains were identified. Similarexperiments were carried out using 3 close structural ana-logues (Figure 3). Syntenic regions (i.e. genomic DNAfragments) containing genes linked from the same locusthat scored highly for dhMotC and at least 1 analogue,compared to an untreated pool of strains, are shown inTable 3. All genomic DNA fragments conferring increasedresistance contained more than 1 gene. To identify indi-vidual genes conferring resistance, the highest-scoringregion for the 2 most potent invasion inhibitors, dhMotCand analogue 20, linking genes AVO1 and ATP19, wasselected, as was the only syntenic region common to allanalogues tested, linking genes SDS22 and ACP1. Eachgene was overexpressed individually and its effect on yeastgrowth in the presence of 30 μM dhMotC was determined.The overexpression of ATP19 (log10 = 0.0142) and ACP1(log10 = 0.0137) conferred a 10-fold and 7-fold growthincrease compared to AVO1 (log10 = 0.0014) and SDS22Table 1: 51 growth-inhibitory compounds identified in halo toxicity screenCompound ρ+ ρ0Antibiotics and antisepticsAntimycin A ++ -Cefoperazone sodium ++ ++Cetylpyridinium chloride + +Chloroxine + +Hexachlorophene ++ ++Myriocin ++ -Thimerosal ++ ++Tunicamycin B ++ ++AnticancerSwainsonine ++ ++Dequalinium analog C-14 linker + +dhMotC ++ -AzolesBifonazole ++ ++Butoconazole nitrate +++ +++Clotrimazole +++ +++Econazole nitrate +++ +++Enilconazole ++ ++Isoconazole +++ +++Ketoconazole ++ ++Miconazole +++ +++Miconazole nitrate +++ +++Oxiconazole nitrate +++ +++Sertaconazole nitrate +++ +++Sulconazole nitrate +++ +++DetergentsCetrimonium bromide + +Digitonin + +FlavonoidsLuteolin ++ ++OtherAdamantamine fumarate ++ ++Amiodarone hydrochloride + +Anthothecol + +Benzalkonium chloride + +Bithionol + +++Cedrelone + +Celastrol + +Dequalinium dichloride ++ ++Elaidylphosphocholine + +Ellagic acid + +Gentian violet ++ ++Miltefosine + +Obtusaquinone + +Phenylmercuric acetate +++ +++Phytosphingosine + +Plumbagin + +Pyrithione zinc ++ ++Pyrvinium pamoate + +Rapamycin +++ +++Shikonin + +SKF96365 ++ ++Suloctidil ++ -Thiram + +Tomatidine hydrochloride + +(log10 = 0.0019) respectively, revealing the genes encod-Totarol + +Page 4 of 17(page number not for citation purposes)BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9ing mitochondrial proteins from each syntenic region asthe suppressors of growth inhibition.Atp19p is a subunit of the mitochondrial F0F1 ATP syn-thase, a large enzyme complex involved in ATP synthesis.This peripheral membrane protein has been proposed tobe involved in the arrangement of the ATP synthase dimerbut it is not required for the formation of enzymaticallyactive ATP synthase and its precise role remains unclear[22]. Acp1p is a mitochondrial matrix acyl carrier proteinthat is involved in fatty acid biosynthesis [23] and its dele-tion causes a respiratory-deficient phenotype. Acp1p isbelieved to be involved in the biosynthesis of octanoate, aprecursor to lipoic acid. Analysis of the genes shown inTable 3 for biological processes showed an enrichment ingenes linked to mitochondrial function (ATP19, ALT1,MRM1, ATP12, MTF2, ACP1, IDP3, YHR162W, CPR3),spanning a wide variety of mitochondrial processesincluding ATP synthase complex assembly, rRNA andmRNA modification and translation, protein folding,NADPH generation, metabolic processes such as fatty acidbeta oxidation and isocitrate metabolism, as well asgenome maintenance. Overall, these results indicate thatincreased mitochondrial function reduces sensitivity todhMotC.To further examine the link between dhMotC sensitivityand mitochondrial function, cells were forced to relyexclusively on mitochondria for ATP production by grow-ing them in glycerol, a nonfermentable carbon source. Ifmitochondrial function is required for dhMotC to killyeast, then growth in these conditions should render yeasthypersensitive to the drug. Indeed, yeast grown in glycerolas the sole carbon source were highly sensitive to 5 μMdhMotC, a concentration that is sub-inhibitory inmedium containing glucose (Figure 4A). P0 cells lackingfunctional mitochondria were completely resistant evento 100 μM dhMotC (Figure 4B). Because functional mito-chondria are not essential for yeast cell survival (ρ0 strainsare viable), these results indicate that dhMotC triggers amitochondria-dependent cell death mechanism.Cell death requires cytochrome c heme lyaseMitochondria have been implicated in programmed celldeath mechanisms in yeast [10]. We next tested a set ofmutants of core players in the mitochondria-dependentdeath response for their sensitivity to dhMotC. Weincluded aif1Δ and mca1Δ, which are both mutants ofimportant mitochondrial cell death effectors, and cyc3Δand the double mutant cyc1Δcyc7Δ [24] which lack maturecytochrome c.Mutants were exposed to 100 μM dhMotC for 24 h andgrowth was compared to untreated controls. Cyc3Δ wasresistant to the compound while aifΔ, mca1Δ andcyc1Δcyc7Δ were strongly inhibited at this high concentra-tion of dhMotC (Figure 5). CYC3 encodes cytochrome cheme lyase, an enzyme catalyzing covalent attachment ofthe heme group to apocytochrome c [25]. While S. cerevi-siae possesses 2 forms of cytochrome c, encoded by CYC1and CYC7 respectively, cyc3Δ mutants lack both holocyto-chromes c. Heme lyase deficiency also prevents mitochon-drial import of the apocytochromes [26].Overexpression of mammalian Bcl-2 can protect fromapoptosis-related death mechanisms in yeast, resulting incell survival [27]. To test whether cells treated withdhMotC could be rescued by Bcl-2, we overexpressedhuman Bcl-2 in yeast cells exposed to the compound.Human Bcl-2 was unable to rescue drug-exposed cells andyeast sensitivity to dhMotC was similar to cells withoutBcl-2 (data not shown).Based on our observations that aif1Δ, mca1Δ andcyc1Δcyc7Δ strains were sensitive to dhMotC and thatdrug-induced cell death could not be rescued by mamma-lian Bcl-2, we assume that these apoptosis-related genesare not directly involved in the death mechanism trig-gered by dhMotC. However, since cyc3Δ was entirelyresistant to the drug, cytochrome c heme lyase plays a spe-cific role in the underlying mitochondrial pathway of celldeath.Chemical-genetic synthetic lethality screen reveals effects of dhMotC on vacuolar pH and vesicle-mediated transportTo further characterize the cellular effects of dhMotC, weconducted a chemical-genetic synthetic lethality screenusing the S. cerevisiae haploid deletion set. In principle,synthetic lethality describes genetic interactions in whichthe combination of 2 nonlethal mutations results inlethality. The method has been applied to identify cellularpathways that "buffer" each other biologically to helpdecipher gene function(s) of individual pathway mem-bers [28]. Global synthetic lethality analysis between nullalleles provides a means to identify genes required forComparison between respiratory-proficient and -deficient yeast strains.Table 1: 51 growth-inhibitory compounds identified in halo toxicity screen (Continued)Table 2: Liquid growth assay for drug sensitivity of ρ+ and ρ0 strainsCompound Growth (% of drug-free assay)ρ+ ρ0Suloctidil (50 μM) 43 98Myriocin (250 nM) 23 93redundant biological processes or functioning in paralleldhMotC (60 μM) 47 100Page 5 of 17(page number not for citation purposes)pathways. In the same way, testing viable mutants forBMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9hypersensitivity to a chemical compound reveals chemi-cal genetic interactions that consist of a set of genes thatbuffer the cell from defects in drug target activity and iden-tifies specific biological processes that are intricatelyinvolved, but are not directly targeted by the drug [7].We screened ~4,700 viable yeast deletion mutants forhypersensitivity to dhMotC by arraying strains onto agarplates containing a sublethal concentration of dhMotCand scoring reduced colony formation. The plates wereincubated at 30°C and colony growth was compared overa period of 4 days. Each mutant was arrayed in duplicateand the screen was carried out twice. Strains displayingincreased sensitivity to dhMotC in both screens are shownin Figure 6. The list of sensitive strains includes 53 nones-sential genes implicated in a variety of biological proc-esses. We found that over 40% of these 53 mutants (22genes, see Figure 6, first column) were either componentsvacuolar assembly and vesicle-based intracellular trans-port.A recent chemical-genetic synthetic lethality screen of over400 small molecules defined a set of multidrug resistance(MDR) genes for deletion strains sensitive to multipledrug treatments [30]. To distinguish between dhMotC-specific and more general cellular drug responses, wecompared the 53 genes to the MDR gene list. None of thegenes involved in the regulation of cellular pH werelabelled as MDR genes, but 6 of 10 genes (60%) involvedin vacuolar assembly and intracellular transport were. Tofurther delineate the cellular response to dhMotC, weasked whether dhMotC directly affected vacuolar pH andintracellular transport.Effect of dhMotC on cellular pHThe chemical-genetic synthetic lethality screen revealedViability test of FY1679-28C/TDEC yeast strain exposed to dhMotCF gure 2Viability test of FY1679-28C/TDEC yeast strain exposed to dhMotC. Short exposure times result in reversible growth inhibition. There is no observable growth after long drug exposure.Page 6 of 17(page number not for citation purposes)of the vacuolar H+-ATPase (V-ATPase) required for theactivity of the proton pump [29], or were implicated in10 of a total of 14 V-ATPase subunits (CUP5, STV1, TFP1,TFP3, VMA2, VMA4, VMA6, VMA7, VMA10, VPH1) and 2BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9membrane proteins required for its assembly (VMA22,VPH2) (Figure 6). This proton pump is a highly conservedmulti-subunit enzyme complex that catalyzes the ATP-driven transport of protons from the cytoplasm to acidicorganelles such as the vacuole and endosomes. As the cen-tral player in organelle acidification in all eukaryotic cells,the pump stores cellular energy in the form of a high con-centration gradient of H+ across organelle-delimitingmembranes, thus constituting a large energy provider forthe cell. Its proton motive force is implicated in a varietyof cellular processes such as protein sorting in the biosyn-thetic and endocytic pathways, proteolytic activation ofzymogen precursors, storage of metabolic buildingblocks, Ca2+ homeostasis, and osmotic control [31].In yeast, cellular pH can be assessed with the lysosomo-tropic amine quinacrine, a basic fluorescent compoundthat accumulates in acidified intracellular compartmentssuch as the vacuole [32]. We used a quinacrine uptakeassay to monitor the pH of vacuoles in dhMotC-treatedyeast. As expected, non-treated cells accumulated quina-crine in the vacuoles, illustrating the acidic nature of theorganelle (Figure 7). However, in cells treated with 60 μMdhMotC, quinacrine staining of the vacuoles could not bedetected, indicating interference of the drug with the V-ATPase. A similar effect was observed with the specific V-ATPase inhibitor concanamycin A (Figure 7). The resultssuggest that dhMotC interferes with vacuolar acidificationthrough the V-ATPase.Structural formulae of dhMotC and close analoguesFigure 3Structural formulae of dhMotC and close analogues.Table 3: Dosage suppressor screenLinked genes\Analogue dhMotC 20 21 27Average log2 fold ratio treated vs. controlARO8 MCM6 3.12 3.41AVO1 ATP19 4.13 2.37GAA1 ALT1 2.13 2.41HYS2 SUI2 YJR008W 2.20 2.43BFR1 MRM1 HIS3 2.01 2.32 2.43MNN11 YJL181W ATP12 PFD1 1.71 1.98MTF2 PRP11 SIR2 2.04 1.72NST1 RHO2 2.03 3.02SDS22 ACP1 3.09 1.71 1.95 2.60SPO1 YNL011C YNL010W IDP3 ASI3 3.88 2.15 3.11YHR162W SOL3 DNA2 2.92 2.99YML081W DUS1 YML079W CPR3 1.75 2.81EBS1 UME6 MSS4 YDR210W 2.35 2.43Page 7 of 17(page number not for citation purposes)Syntenic regions enriched after treatment with motuporamines.BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9We next examined whether dhMotC also affects the acidi-fication of lysosomes in cancer cells. Human MDA-MB-231 breast carcinoma cells were incubated with Lys-oTracker red, a fixable fluorescent dye that accumulates inacidified compartments, treated with DMSO or dhMotC,fixed and examined by fluorescence microscopy. DhMotCcaused a significant decrease in cytoplasmic LysoTrackerred fluorescence intensity compared to DMSO-treatedcontrols (Figure 8). Therefore, dhMotC interferes with lys-osomal acidification in human cells as well as in yeast.Effect of dhMotC on vesicle-mediated transportTo gain additional insight into the involvement of the V-ATPase in the cellular effect of dhMotC and to confirm theresults from the synthetic-genetic lethality screen, wemonitored intracellular trafficking in drug-treated cells.Because a link between the V-ATPase, endocytosis andother transport mechanisms has been shown in severalstudies [31,33], we applied well-established assays tomonitor endocytosis in yeast.First, we followed membrane internalization and vesicle-based transport to the vacuole using FM4-64, a lipophilicstyryl dye that incorporates into the cell membrane, isinternalized and reaches the vacuole through an energy-and temperature-dependent transport mechanism. After90 min in non-treated wild-type yeast cells, FM4-64 wasentirely internalized and labelled the limiting vacuolarmembrane (Figure 9A). Yeast cells treated with 60 μMthe cellular membrane and accumulation of FM4-64 insmall cytoplasmic vesicles (Figure 9A).In a second assay, we monitored the delivery of Luciferyellow (LY), a marker for fluid-phase endocytosis thataccumulates in the vacuolar lumen. LY cannot cross bio-logical membranes and, as a consequence, accumulationin the vacuole depends on vesicular transport. Untreatedyeast cells displayed bright fluorescent staining of the vac-uole by accumulated LY, whereas after 30 min of treat-ment with 60 μM dhMotC, LY failed to enter the cells andcould only be detected as weak staining at the plasmamembrane (Figure 9B). The results from the FM4-64 andLY assays confirm that dhMotC interferes with endocyto-sis.As mentioned, killing of yeast by dhMotC depends on thepresence of functional mitochondria. To test whether thedisruption of endocytosis in drug-treated yeast cells wasalso mitochondria-dependent, we used the FM4-64 assayto monitor endocytosis in ρ0 petite mutants. We observeda disruptive effect of dhMotC on endocytosis in both ρ+and ρ0 cells (data not shown). Based on these results weconcluded that, unlike death induced by dhMotC, inhibi-tion of endocytosis did not require functional mitochon-dria.We next examined whether motuporamines also inhibitintracellular membrane trafficking in cancer cells byHypersensitivity of cells grown on nonfermentable glycerol to dhMotCFigure 4Hypersensitivity of cells grown on nonfermentable glycerol to dhMotC. Growth of respiratory-proficient or -defi-cient yeast (OD600) as function of time in hours in liquid culture under different conditions: Growth in the presence of DMSO (Black diamond) or dhMotC (Black triangle). (A) P+ strain in glycerol with 5 μM dhMotC; (B) P0 strain in glucose with 100 μM dhMotC. Lack of growth of the ρ0 strain in glycerol (Black circle).Page 8 of 17(page number not for citation purposes)dhMotC for 90 min were deficient in vesicle transport tothe vacuole, as shown by residual fluorescent staining atexamining effects on the internalization and degradationof epidermal growth factor (EGF) and its receptor (EGFR).BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9Binding of EGF to EGFR at the plasma membrane leads todimerization of EGFR, stimulation of its tyrosine kinaseactivity and initiation of downstream signaling cascades.The ligand-receptor complex is then downregulated viaendocytosis and intracellular delivery to lysosomes fordegradation [34].MDA-MB-231 cells were incubated with fluorescentlylabelled EGF (FITC-EGF) for 1 h at 4°C, to enable bindingof the ligand to its cell surface receptor. Cells were thenincubated with DMSO or 5 μM motuporamine analogue20 at 37°C for different times and examined by fluores-cence microscopy (Figure 10A). At 4°C, FITC-EGF wasbound to the cell surface. In both DMSO- and analogue20-treated cells, EGF was internalized and showed a simi-lar intracellular distribution for up to 1 h, indicating thatthe compound does not inhibit endocytosis or proteintreated with DMSO, indicating it was degraded in lyso-somes (Figure 10A). In contrast, cells treated with ana-logue 20 showed significantly more cytoplasmic punctateFITC-EGF, indicating that the compound interferes withthe lysosomal delivery and/or degradation of internalizedEGF.To examine the effect of the compound on EGFR localiza-tion, cells were exposed to DMSO or dhMotC and thelocalization of EGFR was determined by immunofluores-cence microscopy. In control cells, EGFR was present atthe plasma membrane, with a noticeable concentration atthe leading edge of migrating cells, as well as in intracellu-lar structures (Figure 10B). In cells treated with dhMotC,EGFR was present in intracellular punctate structures andthere was a clear reduction in plasma membrane-associ-ated EGFR (Figure 10B), indicating that the compounddhMotC sensitivity of haploid strains deleted of cell death-related genesFigure 5dhMotC sensitivity of haploid strains deleted of cell death-related genes. Growth of mutants (OD600) as function of time in hours in YPD liquid culture under 2 different conditions: no drug control DMSO (Black diamond) and 100 μM dhMotC (Black triangle).Page 9 of 17(page number not for citation purposes)transport in the early endocytic pathway. After > 3 h, mostof internalized FITC-EGF had disappeared from cellsinterfered with the lysosomal delivery and/or degradationof internalized EGFR.BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9Page 10 of 17(page number not for citation purposes)53 nonessential genes synthetic lethal with dhMotCFigure 653 nonessential genes synthetic lethal with dhMotC. *: MDR genes as defined in Hillenmeyer et al. [30].DhMotC interferes with vacuolar acidification in yeastFigure 7DhMotC interferes with vacuolar acidification in yeast. Quinacrine staining of yeast under different conditions: Cells were incubated with DMSO, 60 μM dhMotC or 50 μM concanamycin A, stained with the lysosomotropic dye quinacrine and visualized by fluorescence microscopy. Right panel shows control cells in phase contrast microscopy (PC).BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9ConclusionA first screen of differential sensitivity of ρ+ and ρ0 cellsshowed that most drugs, including the therapeutic azoleantifungals, do not require mitochondrial function toexert their growth inhibitory effects. Since ρ0 cells appearincapable of generating ROS [35-38], ROS production bymitochondria is probably not a primary determinant ofthe mechanism of action of most antifungal agents. Only4 chemicals required functional mitochondria to inhibityeast growth. Antimycin A inhibits the transfer of elec-trons to oxygen, resulting in the release of ROS [39].Therefore, the inability of antimycin A to inhibit growthin ρ0 cells can be attributed to the lack of ROS productiondue to the absence of a respiratory chain. Unexpectedly, ρ0cells were also resistant to 3 chemicals that target sphingo-sine and ceramide synthesis. Using dhMotC as an exam-ple, we showed that yeast cell killing requiresholocytochrome c synthase activity. Moreover, forcingcells to rely exclusively on mitochondrial respiration togenerate ATP by growth on a nonfermentable carbonDhMotC interferes with lysosomal acidification in cancer cellsFigure 8DhMotC interferes with lysosomal acidification in cancer cells. Cells were incubated with LysoTracker red followed by DMSO or 5 μM dhMotC, fixed and visualized by fluorescence microscopy. Right panels show nuclear stain.Page 11 of 17(page number not for citation purposes)trons from ubiquinol to the cytochrome bc(1) complex.This inhibition is well known to cause the leakage of elec-source, rendered cells hypersensitive to dhMotC, high-lighting the importance of a functional mitochondrial res-BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9piratory chain activity in its mechanism of action. Theseresults indicate that sphingosine/ceramide biosynthesis isrequired to prevent mitochondria from becoming toxic tocells. In support of this conclusion, it has recently beenshown that ceramide-depleted mitochondria were moresensitive to hydrogen peroxide and ethidium bromide[40] and that ceramide depletion in yeast mitochondria isassociated with programmed cell death and oxidativestress [41].A previous study from our laboratory explored drug-induced haploinsufficiency as a genome-wide approachto study the mechanism of action of drugs [6]. This workidentified sphingosine/ceramide biosynthesis as the vitalpathway inhibited by dhMotC. Interestingly, none of the21 heterozygous mutants showing increased sensitivity todhMotC was deleted of a gene involved in mitochondrialfunction. Therefore, the drug-induced haploinsufficiencyscreen, despite its genome-wide coverage, only partiallyrevealed the mechanism of action of dhMotC, concealinggenes of mitochondrial function involved in the mecha-nism by which dhMotC kills cells.A second screen carried out in the present study, to iden-drial proteins can substantially increase resistance todhMotC, further strengthening the link between mito-chondria and the mechanism of action of the compound.Interestingly, comparing the results from the drug-induced haploinsufficiency screen [6] and the suppressorscreen showed only 1 common gene, SUI2, a subunit ofthe translation initiation factor eIF2 involved in aminoacid starvation [42]. This seemed surprising since thescreens are conceptually similar in that they both rely ongene dose to identify drug-gene interactions. Differencesbetween screens may be related to 1) stoichiometry, e.g.knockdown of 1 subunit of a protein complex is sufficientto reduce its activity and increase drug sensitivity whileoverexpression of 1 subunit of a protein complex is notsufficient to increase its activity and confer resistance, 2)redundancy, i.e. overexpression of a single gene is suffi-cient to confer resistance while knockout of redundantgenes is necessary to detect sensitivity, and 3) unantici-pated technical differences. Alternatively, the results mayindicate a more complex relationship between gene dos-age and drug sensitivity than has been generally consid-ered.The third screen carried out in this study was a chemical-DhMotC interferes with endocytosis in yeastFigure 9DhMotC interferes with endocytosis in yeast. Cells exposed to (A) FM4-64, a fluorescent endocytic marker staining the vacuolar membrane; (B) Lucifer yellow (LY), a fluid-phase endocytic marker accumulating in the vacuole. Cells were incubated with FM4-64 or LY in the presence of DMSO or 60 μM dhMotC and visualized after 90 min chase by fluorescence and phase contrast (PC) microscopy.Page 12 of 17(page number not for citation purposes)tify suppressors of drug sensitivity, clearly showed thatincreasing the expression of genes encoding mitochon-genetic synthetic lethality screen to identify nonessentialgenes that increase sensitivity to dhMotC when com-BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9pletely deleted in haploid strains. 20 of the 53 genes iden-tified have been implicated in resistance to multiple drugsin previous studies [30,43] and were therefore unlikely toprovide clues about the specific mechanism of action ofthe compound. Standing out among the remaining genesare a number involved in the regulation of vacuolar pH,including 10 of 14 V-ATPase subunits and 2 membraneproteins required for V-ATPase assembly. This set of datastrongly implicated vacuolar pH in the mechanism ofaction of dhMotC and led to the demonstration thatdhMotC prevents vacuolar acidification. This effect islikely a consequence of inhibition of sphingosine/cera-mide synthesis by dhMotC, since sphingolipids contain-ing long-chain fatty acids are known to be necessary for V-ATPase activity [44].Chemical-genetic synthetic lethality also revealed a largenumber of genes involved in vacuolar assembly and intra-cellular transport. Further experiments showed thatdhMotC indeed inhibits the delivery of internalized FM4-64 to the vacuole as well as fluid phase endocytosis. Thiseffect is also likely a downstream consequence of inhibi-tion of sphingolipid synthesis since sphingolipids aresynthesis [46]. Defects in vacuolar acidification and endo-cytosis caused by dhMotC occur in ρ0 cells and aretherefore independent of effects on mitochondria.Interestingly, motuporamines also inhibited lysosomeacidification and intracellular trafficking after endocytosisin cancer cells, demonstrating the capacity of thisapproach to predict targets in human cells. These resultsalso provide insight into the mechanism by whichdhMotC inhibits cancer cell invasion. EGF signaling playsan important role in cell migration [47]. Stimulation ofcultured cancer cells with EGF increases invasion andmotility and modulates cell adhesion to extracellularmatrix components in vitro [48] and in vivo [49]. Overex-pression of EGFR causes increased intravasation and lungmetastasis from tumors implanted in the mammary fatpad, and cells overexpressing EGFR are more motile in vivothan adjacent cells not overexpressing EGFR [50]. By inter-fering with vesicle-mediated trafficking of EGFR, motupo-ramines considerably reduce plasma membrane-associated EGFR, and consequently its ability to controlcancer cell migration.Motuporamines inhibit the degradation of internalized FITC-EGF and causes intracellular accumulation of EGFRFigure 10Motuporamines inhibit the degradation of internalized FITC-EGF and causes intracellular accumulation of EGFR. (A) Cells labelled with FITC-EGF at 4°C were exposed to DMSO (control) or 5 μM analogue 20 (motuporamine) for 0, 30 min or 6 h at 37°C, and FITC-EGF was visualized by fluorescence microscopy. (B) Cells were exposed to DMSO (con-trol) or 5 μM analogue 20 for 24 h at 37°C, and EGFR was visualized by immunofluorescence microscopy.Page 13 of 17(page number not for citation purposes)important for protein trafficking [45] and endocytosis isblocked upon interruption of de novo sphingolipid bio-In summary, this study demonstrates the value of usingchemical genomics approaches in Saccharomyces cerevisiaeBMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9to understand the mechanism of action of biologicallyactive chemicals that may have human therapeutic value.However, reliance on a single genome-wide approachmay often provide an incomplete picture of the mecha-nism of action of drugs. Different chemical genomicsscreens can provide complementary information andtheir combined use is probably necessary to provide acomprehensive understanding of the spectrum of differ-ent cellular effects a drug can have on cells.MethodsYeast strains, plasmids and growth conditionsThe haploid set of viable yeast deletion mutants(mat_alpha_041902) was purchased from Invitrogen. Theentire strain collection was used for the chemical-geneticsynthetic lethality screen and individual strains (aifΔ,mca1Δ) were picked for drug sensitivity assays. Non-trans-formed yeast strains were grown in YPD (1% [wt/vol]yeast extract, 2% [wt/vol] bactopeptone and 2% [wt/vol]glucose), or YPgly (2% [vol/vol] glycerol) for media con-taining a nonfermentable carbon source. Respiratory-defi-cient ρ0 strains were generated by inoculating 1 mlsynthetic complete dextrose (SCD) medium (0.67% [wt/vol] yeast nitrogen base without amino acids, 2% [wt/vol]glucose, supplemented with appropriate amino acids)with 10 μl overnight yeast culture (BY4741 or FY1679-28C/TDEC) in the presence of 25 μg/ml filter-sterilizedethidium bromide. After 24 h incubation at 30°C andshaking at 200 rpm, 10 μl of the culture were transferredto 1 ml fresh ethidium bromide-containing SCD medium.After another 24 h shaking at 30°C, 100 μl culture wasplated on YPD agar plates and incubated at 30°C for 2–3days. For overexpression of AVO1, ATP19, SDS22 andACP1, S. cerevisiae FY1679-28C/TDEC cells were trans-formed with GAL1-promoter driven BG1805 containinggene-specific open reading frames (ORFs). Plasmids werepurchased as bacterial stocks from Open Biosystems.Transformed cells were grown in synthetic dropout-GALmedium (0.67% [wt/vol] yeast nitrogen base withoutamino acids, 1% [wt/vol] galactose and 1% [wt/vol] raffi-nose) supplemented with appropriate amino acids. Foroverexpression of mammalian Bcl-2, FY1679-28C/TDECwas transformed with a GAL1-driven pYES-DEST52 con-taining full-length human Bcl-2. Bcl-2 was purchased asan Ultimate™ ORF Clone from Invitrogen and the insertwas transferred to the yeast expression vector through site-specific recombination (Gateway® recombinases, Invitro-gen).Compounds were obtained from the Canadian ChemicalBiology Network Chemical Collection sourced from Prest-wick, Biomol, Sigma and Microsource. Motuporamineswere a generous gift of D. Williams (University of Britishchased from Sigma and solubilised in DMSO. Quinacrinedihydrochloride and Lucifer yellow CH were purchasedfrom Sigma and solubilised in H2O or medium. FM4-64was purchased from Invitrogen.Halo toxicity screenA solution of YPD with 2% agar was prepared by dissolv-ing 5 g of yeast extract, 10 g of peptone and 10 g of agar in450 ml H2O. After autoclaving and cooling to 65°C, 50ml of filter-sterilized 20% glucose solution was added. 45ml of medium were dispensed in Omnitray plates and leftto set. A solution of YPD with 0.5% agar was prepared thesame way by adding 2.5 g agar. For each plate screened, 23ml YPD 0.5% agar were inoculated at 50–55°C with 500μl of an overnight yeast culture (FY1679-28C/TDEC,BY4741 or ρ0 mutants of the same strains) and 22 ml ofthe mixture were poured in the Omnitray plates on top ofthe set YPD 2% agar and left to set for 1 h.Compounds were pin-transferred from 5 mM DMSOstocks in 96-well plates into the cooled agar with a pin-tool robot (BG600, BioRobotics) using 0.7 mm dia. pinsthat deliver 0.34 μl each. Before and between applicationspins were cleaned by submersion in 10% bleach and 70%ethanol for 5 s each followed by drying for 10 s with warmsterile air. The plates were incubated at 30°C for 48 h andhalos were verified by visual inspection.Growth inhibition measurement in liquid cultureYeast strains (OD600 = 0.02) were incubated with appro-priate dilutions of each compound in 200 μl cultures in96-well plates, in addition to DMSO controls. Kineticgrowth curves were generated with a TECAN plate readerby reading the OD every 2 h after agitating the plate priorto reading to suspend the yeast. For growth comparisonsbetween different treatments the exponential part of thegrowth curve was considered and ODs were transformedinto log10 values. The least squares method was applied togenerate a straight line that best fit the data and line slopeswere calculated to compare growth behaviour betweendifferent growth conditions.Drug dosage suppressor screenMulticopy pool construction and growth – A S. cerevisiaerandom genomic library (gift from Martha Cyert) con-structed in a high-copy 2 micron expression vector(YEplac195) with an average insert size of approximately5 kb was transformed into yeast (BY4743) by a standardlithium acetate method [52] and selected in -URA drop-out medium. After 3 days incubation at 30°C, ~106 trans-formants were pooled into medium containing 7%DMSO, aliquoted, and stored at -80°C. For screens, fro-zen aliquots were thawed and inoculated directly into 700Page 14 of 17(page number not for citation purposes)Columbia). They were synthesized as described [51] andsolubilised in DMSO. Myriocin and suloctidil were pur-μl -URA dropout medium to an OD600 = 0.02. Compoundwas added and the pool was grown for 5 generations inBMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/948-well microtiter plates (Nunc). Final compound con-centrations were as follows: 50 μM for dhMotC, analogue20 and 27, 6 μM for analogue 21. An inhibitory concen-tration of at least 50% (IC50) was necessary to provide suf-ficient selection when screens were performed for 5generations. Cells were harvested automatically by a Pack-ard Multiprobe II four-probe liquid-handling system(PerkinElmer). Plasmid isolation, insert PCR amplifica-tion and microarray hybridization – Plasmids were iso-lated using the Zymoprep II plasmid isolation kit (ZymoResearch). The inserts were amplified by PCR with theFailSafe™ PCR System (Epicentre® Biotechnologies) usingcommon M13 primers. PCR cycling conditions were: aninitial melting step at 95°C for 2 min followed by 30cycles at 95°C for 0.5 min, 58°C for 0.5 min and 68°C for10 min followed by a final extension at 68°C for 15 min.The PCR products were purified using the QIAquick PCRpurification kit (Qiagen) and labelled with biotin usingthe BioPrime labelling kit (Invitrogen). Labelled productswere hybridized to Affymetrix TAG4 arrays using the sameprotocols as described for TAG hybridizations [53]. Mult-icopy suppression profiling (MSP) analysis – ORF probeintensities were extracted and normalized. Log2 ratios ofprobes for drug-treated versus DMSO control were calcu-lated. Each ORF was represented by at least 2 probes andthe log2 ratios were averaged to generate a single score foreach gene. To identify each suppressor locus, the log2ratios of intensities were ordered by each ORF's genomiclocation and analyzed using a sliding window to identifyloci that had at least 2 adjacent ORFs with log2 ratios ≥1.6.Quinacrine assayWild type yeast (BY4741) was grown overnight in YPDbuffered with 50 mM NaH2PO4 at pH 7.6. Cells were har-vested by centrifugation (1 min, 13000 rpm, RT, Hereauspico microcentrifuge) and resuspended in 200 μl phos-phate-buffered YPD at OD600 = 0.3. Compounds wereadded and yeast was preincubated for 1 h in the presenceof 60 μM dhMotC or 100 μM concanamycin A. For label-ling with quinacrine, 4 μl of 10 mM stock were added to afinal concentration of 200 μM and the mixture was incu-bated at RT for 5 min. Cells were harvested by centrifuga-tion and washed with SCD medium buffered at pH = 7.6.For visualization yeast cells were resuspended in 10–20 μlbuffered YPD.Yeast endocytosis assaysFor the FM4-64 assay, yeast cells were grown overnightand the cell count was adjusted to OD600 = 1.2. Cells weredivided in 200 μl aliquots and cells were preincubated at30°C in the presence of 60 μM dhMotC or DMSO. Cellswere harvested by centrifugation and resuspended in 10 μland washing with H2O, cells were resuspended in 20 μlYPD in the presence of 60 μM dhMotC or DMSO andincubated at 30°C for 1 1/2 h. To terminate the assay, 1ml of ice-cold 50 mM potassium phosphate buffer con-taining 10 mM NaF and 10 mM NaN3 was added. For vis-ualization, yeast cells were harvested and resuspended in20 μl potassium phosphate buffer. For the Lucifer yellowassay yeast cells were grown to OD600 = 0.1. After harvest-ing by centrifugation the pellet of yeast cells was resus-pended in 90 μl YPD medium and 10 μl of 40 mg/mlLucifer yellow stock was added to a final concentration of4 mg/ml. DhMotC was added immediately to a final con-centration of 60 μM. The mixture was incubated at 30°Cwith shaking at 200 rpm for 1 1/2 h. To stop the assay, 1ml of ice-cold 50 mM potassium phosphate buffer con-taining 10 mM NaF and 10 mM NaN3 was added. Cellswere harvested and washed 3 × with 1 ml ice-cold potas-sium buffer. After the last wash, cells were resuspended in20 μl buffer for visualization. A Zeiss microscope (Axio-vert S100) equipped with filters for epifluorescence andphase contrast was used. Cells stained with quinacrine orLucifer yellow were observed by exciting with 420–490nm light and viewing emitted light with a 520–550 nm fil-ter. Cells stained with FM4-64 were observed by excitingwith 520–550 nm light and viewing emitted light with a610 nm cut-off filter. Photographs were taken with a QIm-aging Microimager II camera. Data were collected as pic-tures of random fields and each field was photographedboth with phase contrast optics and epifluorescence.Cancer cell assaysMDA-MB-231 cells were grown in DMEM/F12 supple-mented with 5% fetal bovine serum and 5 μg/ml insulin.For the LysoTracker red assay, cells grown on coverslipswere incubated with 100 nM LysoTracker red (MolecularProbes) for 25 min before addition of chemicals for 35min. Cells were fixed with 3.7% paraformaldehyde inPBS, washed and DNA was stained with Hoechst 33342.For EGF internalization assays, cells grown on coverslipswere incubated at 4°C for 1 h with 0.4 μg/ml FITC-EGF(Molecular Probes) in cell culture medium supplementedwith 2 mg/ml bovine serum albumin. Cells were thenwashed twice with cold medium before adding chemicalsin cell culture medium at 37°C. After different times at37°C, cells were fixed with 3.7% paraformaldehyde inPBS, washed twice and mounted on slides for microscopy.For EGFR immunostaining, cells grown on coverslips werefixed with 3.7% paraformaldehyde in PBS, permeabilizedwith 0.6% Triton X-100 in PBS, blocked with PBS contain-ing 10% fetal bovine serum and 2% bovine serum albu-min, incubated with 3 μg/ml monoclonal anti-EGFRantibody (Merck), washed and further incubated withCY3-conjugated goat anti-mouse IgG, F(ab') fragment-Page 15 of 17(page number not for citation purposes)YPD. 2 μl of FM4-64 diluted 100 × were added and themixture was incubated on ice for 30 min. After harvestingspecific antibody (Jackson Laboratory).BMC Microbiology 2009, 9:9 http://www.biomedcentral.com/1471-2180/9/9Authors' contributionsDK, LMM, DR, GG, CN, CDR and MR conceived anddesigned the experiments. DK, LMM, SH and DR per-formed the experiments. DK and LMM analyzed the data.DK and MR wrote the paper. All authors read andapproved the final manuscript.AcknowledgementsWe thank Hilary Anderson for fruitful discussions, Martha Cyert for the genomic library, Raymond Andersen and David Williams for motupo-ramines and Philip Hieter for the cyc3Δ yeast deletion strain. CN, GG and SH thank Ron Davis for providing the environment that allowed the devel-opment of the assays they contributed to this study. This work was sup-ported by grants from the Canadian Institute of Health to GG (MOP-81340) and CN (MOP-84305), and by a Canadian Cancer Society grant through the National Cancer Institute of Canada to MR (017392).References1. Sturgeon CM, Kemmer D, Anderson HJ, Roberge M: Yeast as a toolto uncover the cellular targets of drugs. Biotechnol J 2006,1(3):289-298.2. Simon JA, Bedalov A: Yeast as a model system for anticancerdrug discovery. Nat Rev Cancer 2004, 4(6):481-492.3. 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Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis
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