Ploi
Yibmantasiri
,
Peter W.
Bircham
,
David R.
Maass
,
David S.
Bellows
and
Paul H.
Atkinson
*
School of Biological Sciences, Victoria University of Wellington, Room 321, MacDiarmid Building, Kelburn, Wellington, 6012, New Zealand. E-mail: Paul.Atkinson@vuw.ac.nz; Fax: +64 4 463 5331; Tel: +64 4 04 463 7444
First published on 8th November 2013
The pleiotropic drug response (PDR) or multidrug resistance (MDR) are cellular defence mechanisms present in all species to deal with potential toxicity from environmental small molecule toxins or bioactives. The rapid induction of MDR by xenobiotics in mammalian cells and PDR in budding yeast (S. cerevisiae) has been well studied but how pathway specificity is achieved across different structural classes of xenobiotics is not well understood. As a novel approach to this problem we investigated the genome-wide network of genes modulating the yeast PDR. Fluorescently-tagged ABC pumps Pdr5p-GFP and Yor1p-GFP were used as real-time reporters for the Pdr1p/Pdr3p controlled response. Using the yeast non-essential gene deletion set fifty-four gene deletions that suppressed up-regulation of reporter fluorescence to the cell surface in the presence of atorvastatin were identified by high content confocal automated microscopy. Secondary validation using spot dilution assays to known PDR substrates and Western blot assays of Pdr5p expression confirmed 26 genes able to modulate the PDR phenotype. By analysis of network connectivity, an additional 10 genes that fell below the primary screen cut-off were predicted to be involved in PDR and confirmed as above. The PDR modulating genes taken together were enriched in signalling (Rho-GTPase, MAPK), Mediator complexes, and chromatin modification (subunits of ADA and SAGA complexes). Many of the gene deletions cause extra sensitivity in Δpdr1Δpdr3 strains strongly suggesting that there are alternative pathways to upregulate PDR, independently of Pdr1p/Pdr3p. We present here the first high-content microscopy screening for PDR modulators, and identify genes that are previously unsuspected regulators of PDR apparently contributing via network interactions.
This phenomenon is conserved across all kingdoms of life and is a major cause of resistance to antifungal and chemotherapeutic treatments.1–4 The presence of xenobiotics rapidly induces PDR gene expression which in S. cerevisiae is under the control of two homologous transcription factors, Pdr1p and Pdr3p.2,5–7 Although the downstream efflux pumps and targets of Pdr1p, Pdr3p are well annotated, the modulating events between sensing of chemically different xenobiotics and transcriptional activation are less well understood. Whereas xenobiotics may bind directly to Pdr1p/Pdr3p activating them,8 there may be other processes involved. There appears to be a basal generic PDR response activating a common core of 7 genes dependent on Pdr1p/Pdr3p called the early PDR (ePDR). This set of genes is rapidly activated by chemically unrelated xenobiotics (fluphenazine, benomyl). The ePDR acts in combination with additional transcriptional responses leading to drug specific responses.9 In these comparative studies with genome wide microarrays, the authors concluded that even for ePDR there must be “upstream” (of Pdr1p binding) signals mediating the ePDR that are completely unknown. In this current study we investigated PDR-upstream events using functional assays based on the genome-wide yeast deletion set and C-terminus GFP-tagged plasma membrane protein reporters. The intracellular levels and distribution of Pdr5p and Yor1p were investigated using automated high-content confocal microscopy. Both proteins are important ABC transporters under the transcriptional control of Pdr1p/Pdr3p. This allows GFP fluorescence to serve as a sensitive real-time indicator of Pdr1p/Pdr3p transactivation. To show that our reporters respond to xenobiotic treatment cells were treated with atorvastatin, a known PDR substrate. These two transporters were selected as reporters owing to their differences in Pdr1p/Pdr3p dependency2 in which Pdr5p showed more dependency on Pdr1p/Pdr3p than Yor1p. Using proteins with differential Pdr1p/Pdr3p dependency may allow us to identify the major common modulators of the PDR pathway. The design of the assay with our rationale is seen in Fig. 1. We also investigated whether PDR could be affected through network connectivity.
Five screens were carried out, three with the Pdr5p reporter and, due to time constraint, only two with the Yor1p reporter. We scored the common gene deletions affecting, the location of both reporters. A gene deletion was considered as a “hit” if it suppresses plasma membrane location in 2 out of the 3 Pdr5p reporter screens and 1 of the Yor1p reporter screens. The “expression” scores were measured using Acapella Texture analysis software measuring “Ridge”, “Bright”, and “Edge” parameters (Materials and methods). Two out of three of these parameters must show no significant changes between control and atorvastatin treated gene-deletion strains for the gene deletion to be considered as a hit (Materials and methods).
We initially identified 54 gene deletions (“hits”) that prevented the expected increase in Pdr5p and Yor1p expression upon treatment with atorvastatin (Fig. 3, Fig. S2, ESI†). Descriptions for these genes are available in Table S2 (ESI†). Hits appeared in signalling networks (Rho-GTPase, MAPK), RNA polymerase II Mediator complexes, chromatin modification (subunits of SAGA histone acetyltransferase complexes), and sphingophospholipid synthesis.
Gene Ontology (GO) slim-analysis in the function category showed significant enrichment of 11 gene deletions (including PDR1) in transcription activation and co-activation (p-value 0.05 Table S3, ESI†). In addition there was enrichment of deletion mutants that are involved in kinase activity (PSK1, SNF4, KKQ8 and BMH1, (see “Signalling” Fig. 3)), sphingolipid and phospholipid biosynthesis (DPL1, SKN1, PIB2), and mitochondria integrity (PSD1, FMC1, PPE1, FIS1, ATP11, and YME1). Mediator and transcriptional activators such as Gal11p and Ngg1p have previously been shown to regulate or facilitate the PDR response.8,12 However, enrichment of the other transcription factors and activators seen in Fig. 3 in the regulation of PDR1/PDR3 has not previously been described. Such an observation suggests a program of gene transcription is switched on in response to xenobiotics. This is consistent with earlier observations of Fardeau et al. (2007), who suggested signalling pathways upstream of Pdr1p might play a role in the activation of the early PDR response (ePDR).9
To validate the primary screen results, spot dilution assays were carried out with six known PDR substrates (oligomycin, cycloheximide, fluconazole, ketoconazole, atorvastatin, benomyl), according to the description in Experimental methods. Examples of spot dilutions can be seen in Fig. S3 (ESI†). If these genes were important to the cell's survival because of a central role in the PDR response it would be expected that their deletion would show increase PDR-drug sensitivity in all cases. Sub-inhibitory concentrations (not lethal to wild type cells) of the drugs were used. A strain with double deletion of PDR1/PDR3 were used as a positive control, and showed sensitivity to all the compounds used. We found that 17 gene deletions showed an increase in sensitivity toward one or more compounds, whereas only 8 were only sensitive to atorvastatin. Interestingly deletion of PDR1 alone was sufficient to sensitise cells to atorvastatin but none of the other compounds (Table 1). These differential sensitivities further supported existence of an upstream and/or independent pathway of Pdr1p/Pdr3p present in yeast cells.
Gene (ORF) | Sensitivity in Δxxx | Sensitivity in ΔxxxΔpdr1Δpdr3 | Decreased Pdr5p abundance detected by Western blot | Description |
---|---|---|---|---|
“—” means the deletion did not confer sensitivity in spot dilution assay or reduction of Pdr5p in Western blot. Sensitivity observed in ΔxxxΔpdr1Δpdr3 were compared to Δpdr1Δpdr3 control strain. | ||||
BEM2 (YER155C) | Atorvastatin | Atorvastatin | — | Rho GTPase activating protein (RhoGAP) |
Benomyl | Cycloheximide | |||
Cycloheximide | Fluconazole | |||
Ketoconazole | Ketoconazole | |||
Oligomycin | ||||
BMH1 (YER177W) | — | Fluconazole | Yes | 14-3-3 protein regulation of exocytosis, vesicle transport, Ras/MAPK signalling, signalling |
Ketoconazole | ||||
CWH43 (YCR017C) | — | Oligomycin | — | Putative sensor/transporter protein involved in cell wall biogenesis |
DPL1 (YDR294C) | Cycloheximide | — | — | Dihydrosphingosine phosphate lyase, regulates intracellular levels of sphingolipid |
FMC1 (YIL098C) | Cycloheximide | Atorvastatin | — | Required for assembly or stability at high temperature of the F1 sector of mitochondrial F1F0 ATP synthase |
Oligomycin | Fluconazole | |||
Ketoconazole | ||||
Oligomycin | ||||
GCN5 (YGR252W) | — | Atorvastatin | — | Catalytic subunit of the ADA and SAGA histone acetyltransferase complexes |
Cycloheximide | ||||
Fluconazole | ||||
Ketoconazole | ||||
Oligomycin | ||||
GTR2 (YGR163W) | — | Fluconazole | Yes | Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription |
Ketoconazole | ||||
KAP123 (YER110C) | — | Negative genetic interaction | — | Karyopherin beta, mediates nuclear import of ribosomal proteins prior to assembly into ribosomes and import of histones H3 and H4 |
MDM39 (GET1) (YGL020C) | — | Fluconazole | — | Subunit of the GET complex; involved in insertion of proteins into the ER membrane |
Ketoconazole | ||||
OPI3 (YJR073C) | — | Oligomycin | — | Phospholipid methyltransferase catalyzes the last two steps in phosphatidylcholine biosynthesis |
PDR1 (YGL013C) | Atorvastatin | Negative genetic interaction | Yes | Master regulator involved in recruiting other zinc cluster proteins to pleiotropic drug response elements (PDREs) |
PIB2 (YGL023C) | Cycloheximide | Fluconazole | Yes | Protein binding phosphatidylinositol 3-phosphate |
Ketoconazole | Ketoconazole | |||
PIN4 (YBL051C) | Atorvastatin | — | — | G2/M phase progression contains an RNA recognition motif, NLS |
Benomyl | ||||
Cycloheximide | ||||
PSD1 (YNL169C) | Benomyl | — | — | Mitochondrial inner membrane, converts phosphatidylserine to phosphatidylethanolamine |
Ketoconazole | ||||
RCY1 (YJL204C) | Atorvastatin | Negative genetic interaction | — | F-box protein involved in recycling plasma membrane proteins |
Ketoconazole | ||||
RHO4 (YKR055W) | Benomyl | — | — | Small GTPase of the Rho/Rac subfamily of Ras-like proteins |
Cycloheximide | ||||
Fluconazole | ||||
Ketoconazole | ||||
Oligomycin | ||||
ROX3 (YBL093C) | Atorvastatin | Negative genetic interaction | Yes | Subunit of the RNA polymerase II Mediator complex |
Cycloheximide | ||||
Fluconazole | ||||
Ketoconazole | ||||
Oligomycin | ||||
RPN10 (YHR200W) | Cycloheximide | Cycloheximide | — | Non-ATPase base subunit of the 19S regulatory particle (RP) of the 26S proteasome |
Ketoconazole | (Oligomycin?) | |||
SNF4 (YGL155W) | Atorvastatin | Oligomycin | — | Activating gamma subunit of the AMP-activated Snf1p kinase complex |
Oligomycin | ||||
SPT7 (YBR081C) | Cycloheximide | Cycloheximide | Yes | Subunit of the SAGA transcriptional regulatory complex |
Fluconazole | Fluconazole | |||
Ketoconazole | Ketoconazole | |||
Oligomycin | Oligomycin | |||
SRB8 (YCR018W) | Atorvastatin | Atorvastatin | Yes | Subunit of the RNA polymerase II Mediator complex for Pol-II? |
Cycloheximide | Cycloheximide | |||
Fluconazole | ||||
Oligomycin | ||||
SSZ1 (YHR064C) | Cycloheximide | Cycloheximide | — | Hsp70 protein; also involved in pleiotropic drug resistance via activation of PDR1 and PDR5 |
Oligomycin | Fluconazole | |||
Ketoconazole | ||||
Oligomycin | ||||
SWI4 (YER111C) | Cycloheximide | Cycloheximide | — | DNA binding component of the SBF complex (Swi4p–Swi6p), a transcriptional activator |
Fluconazole | ||||
Ketoconazole | ||||
UME6 | Atorvastatin | Atorvastatin | — | Key transcriptional regulator of early meiotic genes |
(YDR207C) | Benomyl | Cycloheximide | ||
Cycloheximide | Fluconazole | |||
Ketoconazole | ||||
Oligomycin | ||||
YME1 (YPR024W) | — | Atorvastatin | — | Mitochondrial inner membrane i-AAA protease complex, responsible for degradation of unfolded or misfolded proteins |
Cycloheximide | ||||
Fluconazole | ||||
Ketoconazole | ||||
Oligomycin |
We found that most genes that caused sensitivity when deleted by themselves also caused extra sensitivity to one or more PDR-substrates when tested in the Δpdr1Δpdr3 background. However, we found that deletion of CWH43, YDR532C, BMH1, GET1, OPI3, GTR2, YME1, and GCN5 only caused increased sensitivity in the Δpdr1Δpdr3 background (Table 1, Fig. S3, ESI†). Deletion of RCY1, KAP123, and ROX3 showed strong negative genetic interaction (no growth) with Δpdr1Δpdr3, while single deletions of these genes individually were viable (Table 1). Interestingly, while single Δrcy1 and Δrox3 mutations increased sensitivity to PDR substrate compounds tested (Table 1), Δkap123 as a single mutation did not cause increased sensitivity in the spot dilution assays. The network of gene deletions that conferred sensitivity in spot dilution assays grouped by GO function is shown in Fig. 4.
The twenty-six gene deletions that increased sensitivity to xenobiotics in the Δpdr1/Δpdr3 background (Table 1) were further subjected to Western blot analysis for Pdr5p (Yor1p was not measured here because no antibody was available). This was to examine if the increase in xenobiotic sensitivity conferred by the deletion was due to decrease in Pdr5p abundance. We found that in addition to Δpdr1 and Δpdr3, several other gene deletions reduced Pdr5p abundance relative to the Pma1p control which showed no alteration in abundance on Western blots. These genes were Δrox3, Δspt7, Δgtr2, and Δbmh1 (Table 1). Since the expression of another plasma membrane protein, Pma1p, an internal control, did not change upon deletion of these genes (Fig. S3 and S4, ESI†), their effect was specific for the PDR response, and not just a global transcriptional activation effect.
Gene | Sensitivity in single deletion | Sensitivity in Δpdr1Δpdr3 background | Decreased Pdr5p abundance detected by Western blot | Description |
---|---|---|---|---|
N/T = not tested. | ||||
ADA2 (YDR448W) | Cycloheximide | Not conclusive | N/T | Transcription coactivator, component of the ADA and SAGA transcriptional adaptor/HAT (histone acetyltransferase) complexes |
DEP1 (YAL013W) | Cycloheximide | Fluconazole | Yes | Transcriptional modulator involved in regulation of structural phospholipid biosynthesis |
Oligomycin | ||||
ERG3 (YLR056W) | Atorvastatin | No | Yes | C-5 sterol desaturase, catalyzes the introduction of a C-5(6) double bond into episterol, a precursor in ergosterol biosynthesis |
Cycloheximide | ||||
Oligomycin | ||||
HEK2 (YBL032W) | No | Ketoconazole | N/T | RNA binding protein |
Oligomycin | ||||
HEX3 (SLX5) (YDL013W) | Atorvastatin | Fluconazole | N/T | Subunit of the Slx5–Slx8 SUMO-targeted ubiquitin ligase (STUbL) complex |
Oligomycin? | Ketoconazole | |||
HSP82 (YPL240C) | N/T | N/T | Yes | Hsp90 chaperone required for pheromone signalling |
LGE1 (YPL055C) | No | No | Yes | Protein of unknown function |
MNN10 (YDR245W) | Atorvastatin | Atorvastatin | N/T | Subunit of a Golgi mannosyltransferase complex |
Benomyl | Cycloheximide | |||
Fluconazole | ||||
Ketoconazole | ||||
NGG1 (YDR176W) | Atorvastatin | N/T | No | Transcriptional regulator; component of transcriptional adaptor and histone acetyltransferase complexes, the ADA complex, the SAGA complex, and the SLIK complex |
Cycloheximide | ||||
Ketoconazole | ||||
Oligomycin | ||||
SPT8 (YLR055C) | Atorvastatin | Fluconazole | Yes | Subunit of the SAGA transcriptional regulatory complex but not present in SAGA-like complex |
Fluconazole | Ketoconazole | |||
Ketoconazole |
Additional genes identified using network interactions predictions were enriched in transcription factors, the SAGA/transcriptional activator and chromatin remodelling (DEP1, NGG1, SPT8, ADA2) as seen by GO analysis.
This result validated the overall screening methodology but also suggested that the PDR phenotypes can be affected, perhaps effected, through genetic networks.
It is possible is that xenobiotics bind directly to Pdr1p.8 Thakur et al. 2008 observed that ketoconazole has a binding KD of 39 μM. However, we found that in wild type cells 20 μM ketoconazole is toxic in YPD agar, i.e. cells are already inviable below the dissociation constant of this putative Pdr1p substrate binding site. It is not known if such direct binding is general for xenobiotics or if it is specific for ketoconazole as shown by Thakur et al. 2008. It is possible Xenobiotic binding alters the conformation of Pdr1p, which then might allow binding of PDR-stress response-specific modifying proteins to bind and activate transcription of PDR-specific genes.13 This view would fit with the interpretations of Fardeau et al. (2007) who proposed that Pdr1p is a stress response factor and a promoter regulator. It has been shown Pdr1p is involved in both basal expression (no xenobiotics), and the rapid upregulation of the PDR genes in the presence of xenobiotics.9
Our study has provided evidence that PDR phenotypes may also be effected through genetic and physical interactions networks. An illustrative example is LGE1, a gene that encodes a protein of unknown function. LGE1 fell below the threshold and was not identified as a “hit” in the primary screen. We hypothesised that deletion of LGE1 might affect the PDR phenotype because LGE1 is synthetic lethal with SWI4, and UME6. As shown in Table 2, deletion of LGE1 did not increase xenobiotic sensitivity in any deletion background. However, it did reduce Pdr5p abundance. This effect might be due to an LGE1 function being associated with the network hubs PDR3, SWI4 and UME6, engaged in transcriptional regulation. By assessing the network, in such a way we may be able to ascertain the pathways in which Lge1p might work.
Overall we observed enrichment of genes involved in signalling in particular those that are involved with Rho-GTPase, Ras, and MAPK. This finding complements our chemical screen in which the GFP labelled ABC-reporters were screened against the 1280 compound Library of Pharmacologically Active Compounds (LOPAC). Several of the compounds that strongly upregulated our ABC-reporters are known to be signalling and kinase inhibitors (Table S4, ESI†). Similar complementary results have been observed in C. albicans. LaFayette et al. (2010) showed that protein kinase inhibitors in a LOPAC screen that have little toxicity on their own, enhanced the toxicity of fluconazole in C. albicans, leading them to propose that kinase signalling regulates multidrug resistance in this pathogenic yeast species.14 These reports combined with the screening results reported here strongly suggest that kinase-signalling pathways are involved in controlling the PDR phenotype.
GO analysis performed on the gene deletions that conferred sensitivity to xenobiotics in the spot dilution assays also showed significant enrichment in genes involved in enzyme activation and protein binding. These genes are BEM2 (a Rho GTPase activating protein), BMH1, and SNF4 (p-value 0.03599). All three genes when deleted individually displayed a decrease in resistance towards many xenobiotics (www.yeastgenome.org). They are also linked with signalling and response to stress (Table 1), especially Bmh1p which controls proteome at post-transcriptional level and involved in many cellular processes from signalling to stress response. Bmh1p and Snf4p are found in plasma membrane, cytoplasm and nucleus and can potentially relay signal from plasma membrane to the nucleus. This adds to the suggestion that a signalling pathway, probably kinase proteins (RAS/MAPK), are required to relay xenobiotic information sensed at the plasma membrane to Pdr1p/Pdr3p in the nucleus. These genes also can also act independently of Pdr1p/Pdr3p (i.e. they all conferred extra sensitivity to strains deleted in Δpdr1Δpdr3), suggesting that they act upstream of Pdr1p/Pdr3p and may also switch on PDR compensatory pathway in the absence of Pdr1p/Pdr3p.
Our assays also revealed additional genes that work independently of PDR1 and PDR3 and contribute to the PDR phenotypes. GO function analysis showed significant enrichment of genes with the SAGA, SWI/SNF chromatin modification and transcriptional activity/co-activation namely UME6, SWI4, SKN7, RSF2, RTG1, ROX3, SRB8, SSN2, and GCN5. We suggest that these genes are likely to be PDR specific modulators and different mediators and chromatin modifications are used to differentiate different stress responses. Some might share the same targets. It is noteworthy that these gene deletions did not confer H2O2 sensitivity, an oxidative stress inducer. Oxidative stress has been linked to PDR target genes.15–19 This result further supports that these genes independently contribute to PDR.
Srb8p has been shown to be linked with the upregulation of PDR5 in cells that have lost the mitochondria genome (ρ0 cells),4 but not in wild-type cells. Our results that showed Srb8p plays a role in normal upregulation of Pdr5p and Yor1p in the presence of xenobiotics suggest that Srb8p might have more roles in the PDR phenotype than is currently known. The SAGA, SWI/SNF chromatin modification complex though often global in function may provide cells with a specific minor backup pathway for Pdr1p/Pdr3p. We propose that in the absence of the major regulator Pdr1p/Pdr3p, these minor regulator components become essential. The importance of minor transcriptional machinery has been proposed by Venter 2009.20 Both SPT8 and SWI4 show negative genetic interactions with GAL11 supporting our proposal that the SAGA and SWI complexes provide cells with an independent alternative pathway to achieve the PDR phenotype. This might be through bypassing the requirement of Gal11p or Pdr1p/Pdr3p altogether. This could explain why the GAL11 deletion does not appear as a hit in our primary screen or spot dilution assay as we anticipated it would. This hypothesis also explains why most of the strains containing deletion of genes in this category were sensitive to xenobiotics and become hypersensitive in Δpdr1Δpdr3 background.
Chromatin remodelling complexes and co-activators, such as RNA polymerase II and Mediator subunits, affect transcriptional activity of gene expression globally.21,22 However, these functions also occur for specific gene expression (“chromatin signalling pathway”).23,24 Chromatin modification/remodelling complexes have been associated with the drug resistant phenotype in S. cerevisiae, C. albicans, and mammalian breast cancer cell lines.25–27 These pathways may provide new targets for overcoming drug resistance, especially if the drug resistant phenotype is achieved from mechanisms other than hyperactivated Pdr1p/Pdr3p. Small molecule inhibitor library screening using such deletion backgrounds and looking for hypersensitivity should provide more insight of how these genes might work together to influence PDR phenotype.27,28
In summary we propose that there is a signalling network of genes upstream of Pdr1p/Pdr3p that conveys the presence of xenobiotics to Pdr1p/Pdr3p in the nucleus inducing a strong and rapid PDR response. These are likely to involve RAS/MAPK signalling pathway(s). We also propose that chromatin remodelling helps cells to achieve a maximum PDR response enhancing Pdr1p/Pdr3p transcriptional activity and/or provide cells with alternative pathways.
Future work will be to analyse the genes that we identified to be involved in PDR signalling. For example we might overexpress genes which show sensitivity in triple deletions with PDR1 PDR3, and see if the over expression rescues the xenobiotic sensitivity phenotype of Δpdr1Δpdr3 strains.
Strain | Genotype | Description |
---|---|---|
BY4741 | MAT a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Wild-type (derived from S288C) |
YCG255 | MAT α, can1Δ::STE2pr-Sp-URA3; lyp1Δ::mCherry-NAT; his3Δ1; leu2Δ0; ura3Δ0; LYS2+ | SGA starting strain |
– mCherry-NAT is red fluorescent protein (RFP) linked with the NAT (nourseothricin) dominant selectable marker under a constitutive promoter. It has a cytoplasmic location | ||
– RFPs act as internal control and as marker for automated cell recognition software | ||
YCG285 | MAT α, can1Δ::STE2pr-URA3; lyp1Δ::mCherry-NAT; PDR5-GFP-HIS3MX6; leu2Δ0 his3Δ1 met15Δ0 ura3Δ0 | Pdr5p reporter strain with C-terminal GFP linked |
YCG334 | MAT α can1Δ::STE2p-URA3; lyp1Δ::mCherry-NAT; YOR1-GFP-HIS3MX6; leu2Δ0 his3Δ1 met15Δ0 ura3Δ0 | Yor1p-GFP reporter strain |
YCG326 | Mat α pdr1Δ::NAT pdr3Δ::URA3 can1Δ::STE2pr_Sphis5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 LYS2+ | Pdr1 Pdr3 knockout |
The method for tetrad dissection was as follows:
Fifty microliters of cells grown in liquid sporulation media for 7 days was spun down at 7000 rpm for 15 seconds, and supernatant was discarded. The cell pellet was re-suspended in 50 μl of zymolyase in sorbitol (0.5 mg ml−1 zymolyase in 1 M sorbitol), and incubated for 10 minutes at 30 °C. Three hundred microliters of sterile distilled water was added to the suspension and the tube was gently inverted to mix. Fifteen microliters of the suspension was spotted onto YPD plates to separate tetrads. Tetrads were dissected using Singer Dissection Microscope.
The dissected spores were grown for 2–3 days at 30 °C, then replica-plated onto selective media using standard procedures.29
All analyses involving measurement of GFP intensity were normalized against the intensity of an internal RFP control.
SGA analysis was carried out according to the method as previously described30 using a Singer RoToR™ HDA bench-top replicator robot (Singer, Somerset, UK) in which the MATα query strain was crossed en masse with the MATa yeast deletion mutant array in 384 colony format. After 2 days incubation at 30 °C final selections plates were then pinned to SC plates in which plates were arrayed so that each gene deletion was duplicated side by side in 384 format. Plates were then incubated at 30 °C overnight, before cells were transferred from colonies to liquid in 384 PekinElmer Cell Carrier plates containing DMSO (control) and drug (atorvastatin 35 μM) in alternate columns. After 4 hours of incubation, cell images were taken using a PerkinElmer Opera® High content microscope. The set up was as follows: 60× Water Immersion lens NA = 1.2 utilising 488 nm and 561 nm lasers with power of 4110 μW and 731 μW respectively. Filter for camera 1 (GFP): 520/35 nm, camera 2 (RFP): 600/40 nm, Detect Dichro: 568, Primary Dichro: 405/488/561/640. Images were captured utilising “binning” of 2 and exposure time of 400 milliseconds. The images were analysed using Acapella™ software to automatically identify cells using RFP cytoplasmic mCherry as described previously.11 Acapella SER Texture feature analysis allowed the observation of subtle intra-cellular changes of biological relevance that could be missed by looking at fluorescence intensity or location alone. SER texture features “Ridge”, “Bright”, and “Edge” were selected due to the fact that they best represented the correct localization of GFP-reporter (Pdr5p, and Yor1p) and gave reproducible results across the different screens.
Yeast cells at a concentration of 5 × 108 cells per ml were serially diluted in water (1:10), and were 2 μl was spotted on YPD plates (unless stated differently) containing drugs of concentration stated in Table 4. Plates were incubated for 2 days at 30 °C and growth phenotype was observed for each strain.
Compound | Concentration used in Δxxx background | Concentration used in Δxxx Δpdr1Δpdr3 background |
---|---|---|
Cycloheximide (CHX) | 0.15 μg ml−1 | 40 nM |
Ketoconazole | 1 μM | 0.5 μM |
Fluconazole | 31 μM | 12 μM |
Oligomycin (in YPGE plate) | 0.35, 0.5 μg ml−1 | 0.35 μg ml−1 |
Benomyl (SC plate) | 60 μM with 25 mM HEPES | Was not used |
Atorvastatin (SC plate) | 35 μM | 5 μM |
After 17 hours, the membrane was blocked with 5% bovine serum albumin (BSA) in 0.1% Tween in Tris-buffered saline (TBST) for 1 hour to reduce non-specific binding. Membrane was then washed with TBST for 10 minutes three times. The membrane was then incubated with a Pdr5p primary antibody (polyclonal Pdr5 (yN-18)) antibody raised in goats (Santa Cruz Co., California, USA stock #SC-27253) diluted 1:500 in 5% TBST for 3 hours at room temperature. The membrane was then washed with TBST for 10 minutes three times before being incubated with the secondary antibody, Donkey polyclonal antigoat antibody labelled with Cy5 obtained from abcam® ab6566 was diluted 1:4000 in 5% TBST for 1 hour in the dark.
The membrane was washed in Tris-buffered saline (TBS) for 10 minutes three times before scanning at 635 nm using Fujifilm FLA-5100 series fluorescent scanner. Pma1p/Pma2p, which are plasma membrane H+-ATPase were used as internal control. Primary antibody for Pma1p/Pma2p raised in rabbits was obtained from Santa Cruz, SC-33735 (diluted at 1:500). Pma1p/Pma2p primary antibody and Cy5 anti-rabbit antibody diluted at 1:4000 were incubated and detected after scanning for Pdr5p.
By establishing genetic networks upstream of PDR1 PDR3, we can derive insights about pathways that modify or compensate the major pathway Pdr1p/Pdr3p-dependent PDR response. Typical of complex gene expression, manipulation of such modifying genes, in principle, will allow observation of specific phenotypes and hence a better understanding of complex drug resistance traits.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mb70351g |
‡ 4.753 is the corrected Z value for multiple testing with equivalent significant level of 0.01. Correction for multiple testing was required because we compared control versus treated for 4500 genes = 4500 multiple tests. The new corrected p-value was 0.01/4500 = 0.000002. |
This journal is © The Royal Society of Chemistry 2014 |