Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0467-6

ORIGINAL PAPER

Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe Kerry A. Rostron . Carole E. Rolph . Clare L. Lawrence

Received: 4 March 2015 / Accepted: 29 April 2015 Ó Springer International Publishing Switzerland 2015

Abstract Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321–328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic K. A. Rostron  C. E. Rolph  C. L. Lawrence (&) School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, UK e-mail: [email protected]

modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses. Keywords Nile red  Schizosaccharomyces pombe  Saccharomyces cerevisiae  Neutral lipids  Phenotype

Introduction Lipids are an important constituent of all eukaryotic cells, which can be broadly classified into neutral and polar lipid subtypes. The more polar phospholipid groups are essential components orchestrating the function and structure of cellular membranes; whereas, neutral lipids specifically triacylglycerols (TAGs) and sterol esters (SEs) are more hydrophobic molecules, holding a central role in the storage of cellular energy (Hutchins et al. 2008). Notably, there is much interest in the neutral storage lipids that form as droplets within cells. Neutral lipid bodies, comprising mainly of TAGs and SEs, are under intense investigation in a variety of research areas. For example, one main area of fungal lipid research pertaining to lipid droplet formation is the production of biofuels. Due to the ease of generating biomass on a large scale, yeast are a convenient alternative to biofuel production from vegetable and plant oils (Santamauro et al. 2014). Furthermore, as demonstrated by the yeasts Yarrowia lipolytica (Y. lipolytica) and Saccharomyces

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cerevisiae (S. cerevisiae), they are amenable to metabolic engineering in order to enhance production and yield of desirable lipid products (Runguphan and Keasling 2014; Tai and Stephanopoulos 2013). Another prominent area of investigation is the mechanisms which regulate lipid metabolism and their impact in human disease. Defects in neutral lipid metabolism have been linked to several pathological states including Alzheimer’s disease, cancer, and diabetes (Di Paolo and Kim 2011; Oresic et al. 2008; Zhang and Du 2012) all of which have global significance. The well characterised yeast species, S. cerevisiae is an attractive model organism to study the regulation of lipid metabolism due to its genetic tractability and large degree of conservation in lipid metabolism to that of higher eukaryotes, as reviewed by Petranovic et al. (2010). A further emerging area of interest is the regulation of lipid metabolism in the fission yeast Schizosaccharomyces pombe (S. pombe) with regards to both human disease research and production of useful industrial products. Little is known about the regulatory pathways of lipid metabolism in S. pombe, and as a result metabolism studies are far less advanced than those in the budding yeast S. cerevisiae (Yazawa et al. 2012; Zhang et al. 2003). The importance of studying lipid metabolism regulatory elements in S. pombe as a model for human disease is great as this species has been demonstrated to have increased similarity to mammalian systems in aspects of stress signalling and cell cycle regulation (Zhang et al. 2003). It is expected that such similarity is mirrored with respect to lipid metabolism as, unlike S. cerevisiae, S. pombe contains homologues to the mammalian transcription factors SREBP-1a (sre1? and sre2?) and SCAP (scp?). Additionally, S. pombe has the potential to be a valuable species in biotechnology as a tool for the production of fatty acids, especially due to its increased production of oleic acid (C18:1), a ricinoleic acid precursor (Yazawa et al. 2012). At present, a published rapid screening method for lipid accumulation in S. pombe has yet to be developed and validated. Such platforms would aid in the rapid identification of superior culture conditions and relevant metabolism regulatory components which can then be promoted for more in depth investigation.

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Lipids begin to accumulate in yeast under conditions of nutrient limitation leading up to the end of logarithmic growth (Beopoulos et al. 2011). Once cells enter the stationary phase of growth they continue to accumulate lipids whilst simultaneously the process of b-oxidation initiates in order to remobilise the stored carbon (Beopoulos et al. 2011). The currently utilised methods to quantify lipids such as gravimetric, gas or liquid chromatography are time consuming and laborious, often involving several steps from extraction to determination of lipid amounts (Bligh and Dyer 1959; Folch et al. 1957; Santamauro et al. 2014; Shahidi 2001). Therefore, screening large numbers of samples is complex, highlighting the need for increased throughput screening methodologies to identify species and strains for further in depth analysis. Further to this, the use of increased throughput assays allows measurements to be taken in situ whilst avoiding the need for multiple extractions, which often necessitate the use of large quantities of environmentally unfriendly organic solvents (Poli et al. 2014). Nile red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is a lipophilic dye that has been used extensively to visualise lipids in a range of organisms. As previously described by Greenspan and Fowler (1985), Nile red has a broad spectrum of fluorescence whereby a shift in emission can be observed which is dependent on the hydrophobicity of the lipid with which it is associated. When associated with more polar lipids a red emission is observed, whilst a yellow-gold emission is observed when Nile red is in the presence of neutral lipids (Greenspan and Fowler 1985). Methodology developed in 2012 by Sitepu et al. (2012) demonstrated that oleaginous and non-oleaginous yeast species could be differentiated by utilising a Nile red assay; however this was applied exclusively to budding, not fission, species of yeast. The ability of this method to reliably detect measurable amounts of fluorescence pertaining to cellular lipid content in low lipid accumulating species, such as S. cerevisiae, highlights that it may additionally be a useful methodology to determine differences in neutral lipid phenotypes between mutant and wild type cells. Previous work validating the use of Nile red assays has been concentrated around determination of lipid accumulation in oleaginous species (Kimura et al. 2004; Sitepu et al. 2012). Phenotypic analyses in non-

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oleaginous yeast species undertaken by employment of Nile red has generally been conducted via means of microscopy, as opposed to assay format (Adeyo et al. 2011; Grimard et al. 2008; Sorger et al. 2004). This study aims to address whether a modified, published methodology for increased throughput assessment of lipids, may be utilised for the fission yeast S. pombe. Further, this study sought to address, for the first time, the capability of the current method to determine lipid phenotypes in both S. cerevisiae and S. pombe deletion strains.

Materials and methods Yeast strains and culture conditions Saccharomyces cerevisiae and S. pombe strains utilised in this study are listed in Table 1. Yeast were pre-cultured in appropriate complex medium, either YPD [2 % glucose, 1 % peptone, 1 % yeast extract] or YES [3 % glucose, 0.5 % yeast extract, 0.005 % amino acids leucine, lysine, adenine, histidine and uracil] (ForMediumTM, UK) before resuspending in defined minimal medium YNB (S. cerevisiae) [0.67 % yeast nitrogen base without amino acids (ForMediumTM, UK) 2 % glucose and 0.002 %, appropriate amino acids except for threonine, uracil, tyrosine, lysine at 0.001 % and 0.005 % leucine] or Edinburgh Minimal Media (EMM) [1.2 % EMM without dextrose and amino acids (ForMediumTM UK), 2 % glucose and 0.0225 % appropriate amino acids). Yeast cells were resuspended at a cell density of *2.0 9 106 cells/ml (corresponding to an OD595nm * 0.1). Cultures were incubated at 30 °C, with shaking at 180 rpm until desired growth phases were reached.

Fluorescence microscopy Cells for microscopy were stained by addition of 5 lg/ ml Nile red in acetone per 250 ll of yeast culture before incubation for 5 min in the dark at room temperature. Stained cultures were then centrifuged for 2 min at 492 x g before washing twice in phosphate buffered saline (PBS) (Fisher, UK). Cells were resuspended in 10 % (v/v) formalin and fixed for 15 min in the dark at room temperature before centrifugation and a further 2 washes with PBS. Cell pellets were resuspended in SlowFade (Invitrogen, UK). Nile red stained cells were viewed on an AxioObserver Z1 inverted microscope using an Axiocam MRm imaging system with monochrome camera. Lipid distribution was visualised using filters for Ds Red (Ex 545/Em 572 nm) and EGFP (Ex 488/Em 509 nm). Nile red screening Methodology was carried out according to a modified Sitepu et al. procedure (Sitepu et al. 2012). Briefly, cells were harvested by centrifugation at 4929g for 2 min. In order to remove the high background fluorescence of the medium, the resultant cell pellet was washed twice in PBS (Fisher, UK) before being resuspended in PBS at a density of 2 9 107 cells/ml (OD 595 nm * 1.0) determined using a haemocytometer. Optical density, 595 nm, was recorded again after final resuspension and *5 9 106 cells (250 ll) were aliquoted into the wells of a black, clear bottomed 96-well plate (Krystal 2000, Porvair Sciences) in triplicate. A 25 ll volume of DMSO/ PBS (1:1 v/v) was added to each well before addition of 25 ll 60 lg/ml Nile red in acetone to a final concentration of 5 lg/ml per well. The wavelengths

Table 1 Yeast strains utilised in the present study Yeast species

Strain

Genotype

Source

S. cerevisiae

BY4741

MATa his3D1 leu2D0 met15D0 ura3D0

Brachmann et al.

S. cerevisiae

Y02501

MATa dga1::kan his3D1 leu2D0 met15D0 ura3D0

Euroscarf

S. cerevisiae

Y05383

MATa lro1::kan his3D1 leu2D0 met15D0 ura3D0

Euroscarf

S. cerevisiae

BY5648

MATa fat1::HIS3 ade2-1 leu2-1,112 ura3-1 trp1-1 his3-11,15

NBRP of the MEXT, Japan

S. pombe

CLL001

h?ura4-D18 leu1-32 ade6-M210 his7-366

Laboratory stock

S. pombe

BG_4249

h? dga1:: kan ura4-D18 leu1-32 ade6-M210 his7-366

Bioneer Corporation

S. pombe

BG_H1649

h? ptl3::kan ura4-D18 leu1-32 ade6-M210 his7-366

Bioneer Corporation

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excitation 485 nm emission 535 nm were utilised to determine relative neutral lipid amounts in the samples. The number of plate reader cycles was optimised by reading samples on a kinetic reading every 60 s for 20 min. The cycle before saturation point of the Nile red dye, where fluorescence signal reached a plateau, was determined and subsequently used for further screens in all cases. Plates were read immediately after addition of Nile red using the determined kinetic cycle on a Tecan Genios Pro platereader using the top 50 % mirror setting. The gain settings for each wavelength of individual experiments was set to optimal whereby the gain value was determined by the plate reader. The determined gain setting was recorded after the initial screen and set manually to the same values for further screens conducted within the triplicate. Data was corrected against PBS containing 1/10 volume DMSO/PBS (1:1 v/v) and 5 lg/ml Nile red in acetone and fluorescence further normalised using the optical density reading of each sample. In order to compile data generated from separate Nile red (NR) assays within the triplicate, the highest average background fluorescence was divided by the lower average background fluorescence to give a ratio. Fluorescence values of the samples were then adjusted to the assay with the highest background fluorescence by multiplying fluorescence values for each sample by the calculated ratio. Statistics All data is displayed as means of triplicate experiments ± SD. Statistical analyses were performed by means of one way ANOVA with Tukey post hoc comparisons using GraphPad Prism 5.0 software. A significant result was determined to have a p value of \0.05.

data has demonstrated acquisition of neutral lipid data from a variety of excitation and emission wavelengths including 485/535 nm (Dey et al. 2014) and 535/590 nm (Sitepu et al. 2012). As such, fluorescence microscopy was conducted on stationary phase S. cerevisiae and S. pombe cells to determine the most suitable wavelengths for detection of neutral lipids. Figure 1 shows fluorescence of Nile red stained S. cerevisiae (1a) and S. pombe (1b) in the stationary phase of growth. As shown in Fig. 1, when viewed under green fluorescence (EGFP filter Ex. 488/Em. 509 nm) stationary phase NR stained S. cerevisiae and S. pombe cells exhibited discrete fluorescent bodies, of varying sizes, throughout the cytoplasm of the cell. This pattern of fluorescence was similar across the population of yeast cells observed and indicated the presence of intracellular lipid droplets. However, under red fluorescence (DsRed filter Ex. 545/Em. 572 nm), S. cerevisiae cells showed fluorescent bodies within the cytoplasm in combination with consistent staining across the cell. This suggests that, in addition to neutral lipids, this wavelength also detects polar lipids present in cellular membranous structures and in the lipid monolayer surrounding the lipid droplets (Beller et al. 2010). This is consistent with data presented by Greenspan and Fowler (1985). In the case of S. pombe cells viewed under red fluorescence (DsRed filter Ex. 545/Em. 572 nm), limited staining indicative of intracellular lipid droplets was observed, with fluorescence output appearing almost exclusive to membranous structures. This highlights that Nile red could be potentially applied to fission yeast in order to additionally investigate carbon flux changes of more polar lipid subtypes. The efficacy of Nile red to determine changes in the polar lipid pool by utilising red emission would however require further investigation and validation compared with more advanced chromatographic analyses.

Results and discussion Detection of neutral lipid droplets by fluorescence microscopy of Nile red stained budding and fission yeast species It has been demonstrated that Nile red exhibits a wide spectrum of fluorescence which is dependent on the hydrophobicity of the lipid with which it is associated (Greenspan and Fowler 1985). Previously published

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Determination of neutral lipid content in wild type and mutant strains of the non-oleaginous budding yeast, S. cerevisiae As with oleaginous yeast species, S. cerevisiae cells accumulate lipid as they enter the stationary phase of growth (Sandager et al. 2002). However, as a nonoleaginous yeast it accumulates lipid up to approximately 11 % of the cellular dry weight

Antonie van Leeuwenhoek

509 nm Emission

572 nm Emission

Merged

Overlay

(a)

(b)

Fig. 1 Fluorescence microscopy of Nile red stained S. cerevisiae and S. pombe cells, 1009 magnification, scale 5 lm. a Nile red stained S. cerevisiae in the stationary phase of growth viewed with filter sets EGFP (Ex. 488/Em. 509 nm) and DsRed (Ex. 545/Em 572 nm). b Nile red stained S. pombe cells in the

stationary phase of growth viewed with filter sets EGFP (Ex. 488/Em. 509 nm) and DsRed (Ex. 545/Em 572 nm). Merged: Fluorescence from both EGFP and DsRed to shown colocalisation of fluorescence signal. Overlay: Merged fluorescence images with bright field image of cells

(Kalscheuer et al. 2004) in comparison to oleaginous species such as Lipomyces starkeyi (L. starkeyi), which can accumulate lipids up to 70 % of its dry cell mass in stationary phase. The low lipid content of S. cerevisiae cells means accurate measurement by gravimetric means is difficult (unpublished data), therefore alternative methods are required. The Nile red assay utilised was adapted from the method by Sitepu et al. (2012) with a number of modifications. To undertake screening, Krystal 2000 96 well plates were chosen over plates utilised in the previous study (Costar, product #3370). This was due to consistency of individually moulded wells, thus increasing specificity of well fluorescent readings by avoidance of lateral piping of light. Although it has been previously described that Nile red has varying fluorescence properties in a range of media types (Sitepu et al. 2012), the fluorescence properties of spent culture media was not determined. As such, all cells were washed twice in PBS to reduce background fluorescence resulting from culture medium. Fluorescence data obtained was corrected against not only cell

density, but also background fluorescence resulting from PBS. This ensured any changes in relative fluorescence unit (RFU) values were the result of cellular lipid content alone. To establish growth phases on which to conduct Nile red screening for phenotypic analysis in stationary phase S. cerevisiae cultures, a time course was conducted to identify fluorescence amounts pertaining to lipid content in different phases of growth. After an initial lag phase, cells typically entered exponential growth at 5 h post resuspension in YNB, exponential growth then continued until early stationary phase at 12 h, Fig. 2a. From 12 to 72 h little additional growth of S. cerevisiae was observed via optical density (data not shown). As shown in Fig. 2b, fluorescence corresponding to neutral lipids increased over time in S. cerevisiae cells from exponential growth into stationary phase at 18 h. As expected maximal neutral lipid accumulation was observed when cells reached mid stationary phase (24 h) before a decrease was observed at late stationary phase, (48 h onwards), which is likely to be observed due to remobilisation of stored lipid as a

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(a)

(b)

2.5 Culture 1 Culture 2

OD595nm

2.0

1.5

1.0

0.5 0.0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time hrs

(c)

Fig. 2 a Growth curve of S. cerevisiae in YNB medium. Cells from log phase YPD cultures were resuspended in minimal medium. Cultures were allowed to grow, with shaking at 180 rpm 30 °C and readings taken as seen appropriate until stationary phase was reached, as indicated by plateaued growth. b Time course of neutral lipid fluorescence intensity measured by Nile red with growth of wild type S. cerevisiae. c Neutral lipid fluorescence intensity of Nile red stained S. cerevisiae wild

type, dga1D, lro1D and fat1D mutants in early exponential and stationary phase of growth using excitation and emission wavelengths of 485/20 nm and 535/25 nm respectively. Data shown as means of triplicates ± SD. Significance indicated between exponential and stationary phases ***p B 0.001 and difference in stationary phase between strains indicated by solid black lines   p B 0.01 and    p B 0.001. Data analysed by one way ANOVA with Tukey post hoc test

consequence of beta oxidation (Beopoulos et al. 2011; Beopoulos et al. 2008). In order to test the ability and sensitivity of the Nile red method to detect neutral lipid phenotypes, a screen was undertaken with characterised S. cerevisiae gene deletions known to produce a predominantly neutral lipid phenotype. The deletions used were dga1, lro1 and fat1. The genes dga1 and lro1 encode enzymes involved in triacylglycerol synthesis with Dga1p catalysing the terminal step by acylating diacylglycerol using acyl-CoA as an acyl donor (Oelkers et al. 2002). Lro1p is an acyltransferase that catalyses diacylglycerol esterification (Oelkers et al. 2000). In

S. cerevisiae, Dga1p and Lro1p catalyse 96 % of TAG synthesis (Sandager et al. 2002) so cells deleted for these genes would be expected to display a decreased neutral lipid phenotype compared with wild type cells. fat1 encodes a protein, which functions as fatty acid transporter and a very long chain acyl-CoA synthetase (Choi and Martin 1999); its deletion could be expected to result in an increased intracellular lipid phenotype (Watkins et al. 1998) (Fig. 3). Growth curves were undertaken on all strains used to ensure growth was similar between the mutant and wild type cells. No observed growth defects were seen with the deletions tested (data not shown). As such, the Nile red assay

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was carried out utilising stationary phase (18 h) cells to infer phenotype along with cells in early exponential phase (5 h) as a low lipid control. Figure 2c shows the results of the neutral lipid fluorescence intensity of wild type and deletion strains in both early exponential and stationary phases of growth. Consistent with previous observations (Oelkers et al. 2002; Sandager et al. 2002), Fig. 2c shows that cells deficient in dga1 exhibited a significant reduction in relative fluorescence attributable to neutral lipids compared to that of wild type cells with a 1.4 fold decrease. This supports findings by Oelkers et al., who demonstrated a decrease (1.9 fold) in the amount of triacylglycerol synthesis via metabolic labelling (Oelkers et al. 2002). Also consistent with the findings of Oelkers et al, cells lacking lro1 showed a slight decrease in neutral lipids in stationary phase that failed to reach statistical significance. Cells deleted for fat1, as expected, exhibited a significant increase in relative fluorescence levels compared to wild type suggesting an accumulation of hydrophobic very long chain fatty acids (Watkins et al. 1998). These results demonstrate that the modified Nile red method developed by Sitepu et al. 2012 is sensitive enough to detect neutral lipid changes in S. cerevisiae mutant cells. Nile red can determine neutral lipid content in the non-oleaginous fission yeast, S. pombe and additionally detect neutral lipid phenotypes in mutant strains Little research has been undertaken on lipid accumulation in the fission yeast S. pombe. However, due to its

potential importance in both biotechnology and the study of human disease, we extended our study to examine the use of the Nile red assay to examine the neutral lipid content of S. pombe cells. As limited work has been published that describes lipid accumulation in S. pombe cells, growth phases in which to undertake Nile red screening were examined. This was carried out via measurement of fluorescence pertaining to relative neutral lipid amounts, utilising the Nile red assay, over the course of S. pombe growth. After an initial lag phase, S. pombe cells typically entered exponential growth at 8 h post resuspension in EMM, exponential growth then continued until early stationary phase at 24 h, Fig. 4a. From 24 to 72 h little additional growth of S. cerevisiae was observed via optical density (data not shown). Interestingly, unlike S. cerevisiae, we found a higher relative neutral lipid content existed in exponentially growing S. pombe cells (8 h) compared to those in late exponential (18 h) and early stationary phase (24 h), Fig. 4b. Two observations could explain this result. Firstly, as S. pombe has a polarised manner of growth, dividing via medial fission, increased levels of sterols are present at the tips during exponential growth before accumulating at the completed septum closer to cell division (Wachtler et al. 2003). Secondly, intracellular lipid droplets in S. pombe are increased in the G2 phase of the cell cycle (Long et al. 2012). The increased amount of neutral lipids observed in S. pombe during exponential growth could have important implications for both biotechnology and human disease research. Firstly, in human disease DAG

LP

PA

Glycolysis

Dga

TAG

DAG Lipases

Lro

FFA

Fat1

TAG

Acetyl-CoA

Fig. 3 Simplified overview of neutral lipid synthesis in S. cerevisiae, highlighting positions of Dga1, Lro1 and Fat1 which were utilised to validate the Nile red assay in the present study.

LPA lysophosphatidic acid, PA phosphatidic acid, TAG triacylglycerol, DAG diacylglycerol, FFA free fatty acids. Adapted from Kohlwein et al. (2013)

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(b)

(a) 4.0 Culture 1 Culture 2

3.5

OD595nm

3.0 2.5 2.0 1.5 1.0 0.5 0.0

0

5

10

15

20

25

30

35

Time hrs

(c)

Fig. 4 a Growth curve of S. pombe in EMM medium. Cells from log phase YES cultures were resuspended in minimal medium. Cultures were allowed to grow, with shaking at 180 rpm 30 °C and readings taken as seen appropriate until stationary phase was reached, as indicated by plateaued growth. b Time course of neutral lipid fluorescence intensity measured by Nile red with growth of wild type S. pombe. c Neutral lipid

fluorescence intensity of Nile red stained S. pombe wild type, dga1D, lro1D and fat1D mutants in stationary phase of growth using excitation and emission wavelengths of 485/20 nm and 535/25 nm respectively. Data shown as means of triplicates ± SD. Significance indicated in stationary phase between strains indicated by solid black lines  p B 0.05 and   p B 0.01. Data analysed by one way ANOVA with Tukey post hoc test

research, S. pombe may prove to be a potentially useful organism for modelling aberrant lipid accumulation in cancer. As proliferating cancerous cells undergo a high rate of de novo lipid accumulation (Danie¨ls et al. 2014), the increased levels of neutral lipid accumulation seen in active exponential division in S. pombe could be useful for deconstructing the regulation of increased de novo lipid formation. This is in contrast to S. cerevisiae, whereby a gradual increase is seen throughout exponential growth with maximal accumulation observed in stationary phase where cells have ceased dividing. Secondly in biotechnology, increased lipid accumulation in exponential phase may allow the use of continuous culture, rather than a

batch process, to harvest important lipid products. This would have several advantages including an earlier and steady production of lipids, lower capital and fixed costs, better control of the system and longer running time once the culture has been established. This observation may also be useful if the molecular basis of increased lipid production in the exponential growth phase were known. This could serve to aid in the metabolic engineering of S. pombe, in order to maximise lipid accumulation with increased biomass in the stationary phase of growth. To assess whether the Nile red assay could be also be applied to S. pombe cells to determine neutral lipid phenotypes, Nile red screening was conducted

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utilising S. pombe deletion strains expected to deliver a predominantly neutral lipid phenotype. The S. pombe dga1D mutant, analogous to its S. cerevisiae counterpart, is expected to show a significant decrease in relative neutral lipid amount. The deletion of ptl3, a predicted triacylglycerol lipase, is expected to demonstrate an increased relative quantity of neutral lipid. Unlike S. cerevisiae, a low lipid exponentially growing control could not be employed for S. pombe screening; as such S. pombe mutant cells were screened during early stationary phase at 24 h and compared against wild type cells. As shown in Fig. 4c, cells deleted for dga1 exhibited a 1.5 fold decrease in fluorescence attributed to neutral lipids when compared to the wild type. This is similar to the impact on neutral lipid levels seen in the S. cerevisiae dga1 deletion and confirms the hypothesis that S. pombe dga1 has a role mainly in stationary phase cells (Zhang et al. 2003). Cells lacking ptl3 displayed a 1.4 fold increase in neutral lipid fluorescence compared to wild type, consistent with recently published gas chromatography data where a 1.7 fold increase in average cellular TAG amounts occurred as a result of ptl3 deletion (Yazawa et al. 2012). Due to their extensive characterisation, both S. cerevisiae and S. pombe, serve as excellent model organisms in eukaryl biology. This, coupled with a plethora of available genetically modified strains, means that novel lipogenic regulatory mechanisms can be identified. This may provide further insight into aberrant metabolism in human disease processes, or can be utilised to increase production and yield of products of biotechnological importance. From our own unpublished data, using the Nile red assay described in this study, we have identified two components of a major signalling pathway that have not been previously implicated in neutral lipid biosynthesis. This study demonstrates, for the first time, that a Nile red assay can be applied to the fission yeast S. pombe to infer relative neutral lipid amounts and detect neutral lipid phenotypes. This is of particular importance due to growing interest and research in the area of S. pombe lipid metabolism. However, this method is not without its limitations. The method is limited to the general pool of neutral lipid, therefore further analysis to determine changes in molecular species would need to be conducted by other means. In addition, it may not be sensitive

enough to detect changes in relative lipid amounts when only a slight phenotype results. However, the rapid nature, repeatability and potential scalability of this methodology means that interesting yeast strains, species and indeed culture conditions, that have significant impact on lipid amounts, can be readily segregated for further analysis by such methods as gas chromatography. This ultimately means avoiding time consuming, labour intensive analyses of samples that may not yield a desirable lipid phenotype.

Conclusions Utilising the Nile red assay for phenotypic characterisation of yeast lipid content avoids the requirement for time consuming, labour intensive sample analyses which may not yield a desirable phenotype. The rapid nature, scalability and repeatability of this method means that strains harbouring genetic modifications, having significant impact on relative lipid amounts, can be readily segregated for further in depth analyses. In addition, we have demonstrated and validated, for the first time, its use in characterising neutral lipid content in wild type and deletion strains of the low lipid non-oleaginous fission yeast S. pombe. Acknowledgments This work is supported by the University of Central Lancashire and Brain Tumour North West. We acknowledge Bioneer Corporation and NBRP of the MEXT, Japan for providing deletion strains used in this study. We thank Dr. Vicky Jones and Dr. Gail Welsby for support with microscopy, Mr. Tony Dickson for technical assistance, Dr. Christopher Smith for support with the statistical analysis and Dr. Stephen Lawrence for critically reading the manuscript. Conflict of interest

None.

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Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. Th...
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