© 2016. Published by The Company of Biologists Ltd.
Targeting surface voids to counter membrane disorders in lipointoxicationrelated diseases
Romain Ferru-Clément1,2, Miroslava Spanova1, Shalinee Dhayal3, Noel G. Morgan3, Reynald Hélye2,
Frédéric Becq1, Hisaaki Hirose4, Bruno Antonny4, Lydie Vamparys5, Patrick F.J. Fuchs5 and Thierry
Ferreira1
1
Laboratoire « Signalisation et Transports Ioniques Membranaires (STIM) », CNRS ERL 7368, Université de Poitiers, 1, rue
Georges Bonnet 86073 Poitiers Cedex 9, France
2
Société d’Accélération du Transfert de Technologie (SATT) Grand Centre, 8 rue Pablo Picasso, 63000 Clermont-Ferrand,
France
3
Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter EX2 5DW, UK
4Institut
de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 7275, Université de Nice Sofia-Antipolis, 660 route des
Lucioles, 06560 Valbonne, France
5
« Dynamique des membranes et trafic intracellulaire », Institut Jacques Monod, CNRS UMR 7592, Université Paris
Diderot, Sorbonne Paris Cité, 15 rue Hélène Brion, 75013 Paris, France
Correspondence should be addressed to TF (e.mail: [email protected])
Abstract
Saturated fatty acids (SFA), which are abundant in the so-called Western-diet, have been shown to
efficiently incorporate within membrane phospholipids (PL) and therefore impact organelle integrity
and function in many cell-types. In the present study, we have developed a yeast-based two-step
assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated
lipointoxication. The compounds identified here were effective in relieving lipointoxication in
molecular dynamics simulations on bilayers revealed that these molecules, albeit according to
different mechanisms, can generate voids at the membrane surface. The resulting surface defects
correlate with the recruitment of loose lipid packing/void-sensing proteins required for vesicular
budding, a central cellular process that is precluded under SFA-accumulation. Altogether, the results
presented here point at modulation of surface voids as a central parameter to consider in order to
counter the impacts of SFA on cell function.
JCS Advance Online Article. Posted on 3 May 2016
Journal of Cell Science • Advance article
mammalian β-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and
Introduction
Eukaryotic cells are sub-divided into highly specialized organelles, thereby allowing specific
functions to occur efficiently and simultaneously in different compartments of the cell. To allow the
optimal function of these subcellular compartments, cells have developed complex and highly
regulated processes to maintain specific phospholipid (PL) compositions within membranes, such as
PL remodeling (Yamashita et al., 2014), to regulate the acyl chain composition, and selective lipid
transport from their site of synthesis to the accepting membrane (Gillon et al., 2012; Tamura et al.,
2014). As a consequence, when a single PL type is considered, its acyl chain composition varies
greatly depending on the organelle: the saturation rate of the acyl chains increases progressively
along the secretory pathway to reach its maximum at the Plasma Membrane (PM) (Schneiter et al.,
1999). The fatty acyl content of a PL molecule contributes to its overall shape, which is defined by
the cross-sectional area of the headgroup relative to the cross-sectional area of its acyl chains (de
Kroon, 2007). For example, an oleoyl chain (C18:1) occupies a larger volume than a palmitoyl chain
(C16:0), because the double bond induces a “kink” in the middle of the chain (Deguil et al., 2011).
Therefore, inserting a kinked acyl chain within a PL species tends to shift its shape from rather
cylindrical to conical. Conical lipids have major impacts on the biophysical properties of biological
membranes: when inserted into a bilayer they tend to generate voids at the membrane surface
(Vamparys et al., 2013).
Antonny to propose the coexistence of two broad territories within the cell (Bigay and Antonny,
2012): the territory of loose lipid packing/large and deep surface voids (i. e. from the Endoplasmic
Reticulum (ER) to the trans-Golgi Network (TGN)), where weakly charged monounsaturated PL are
overrepresented, and the territory of electrostatics, which is defined by the presence of saturated,
highly charged lipids (i. e. PM and endosomal compartments). In the territory of large surface voids,
cytosolic proteins take advantage of the surface clefts generated by the membrane packing defects
Journal of Cell Science • Advance article
The heterogeneity in the fatty acyl composition of PL along the secretory pathway led Bigay and
to be recruited to the organelle membranes, and thereby regulate central cellular processes, such as
vesicular budding (Bigay and Antonny, 2012; Bigay et al., 2005).
Not surprisingly, perturbations of the fatty acid composition of PL have dramatic effects of
organelle function. For example, exogenously supplied saturated fatty acids (SFAs), which efficiently
incorporate within PL, have major impacts on the integrity of the organelles from the loose-lipid
packing territory. Early in the secretory pathway, SFA have been shown to alter vesicular budding in
the ER, promote the accumulation of unfolded proteins within this compartment and induce a socalled ER stress, which can ultimately lead to cell death by apoptosis (Borradaile et al., 2006; Dhayal
and Morgan, 2011; Guo et al., 2007; Gwiazda et al., 2009; Laybutt et al., 2007; Pineau et al., 2009;
Preston et al., 2009). Later in the pathway, SFAs have also been demonstrated to affect vesicle
formation at the Golgi in a process that could be directly connected to increased lipid-packing (Payet
et al., 2013). Indeed, we demonstrated that saturated PL alter the recruitment of proteins of the
ArfGAP family to nascent vesicles in vivo, which emerge from this compartment, both in yeast and
human cells (Payet et al., 2013; Vanni et al., 2014). Since ArfGAP and mechanistically-related
proteins are required for vesicle transport and contain motifs that sense lipid packing (Bigay et al.,
2005), it was proposed that this process could account for the general disruption of the secretory
pathway under SFA-accumulation (Payet et al., 2013).
Such observations could be very pertinent for understanding several physiopathological states in
etiology of several high-occurrence “way-of-life” related diseases, including Type-2 diabetes, hepatic
steatosis and atherosclerosis (Akazawa et al., 2010; Alkhateeb et al., 2007; Cnop et al., 2001; Miller
et al., 2005).
In the present study, we isolated new molecules able to counter SFA-related lipointoxication.
Altogether, the results presented here validate the method as an efficient high-throughput screening
approach to identify new lipointoxication inhibitors. Moreover, they point at modulation of surface
Journal of Cell Science • Advance article
human. Indeed, SFA, which are abundant in the Western-diet, have been directly related to the
voids as a central parameter to consider in order to efficiently counter the impacts of SFA on cell
function in certain human diseases.
Results
A two-step screening strategy to identify molecules that relieve lipointoxication
The first screening step used in this study relies on a knock-out mutant of the Δ–aminolevulinate
(δ-ALA) synthase (hem1Δ), which brings together most of the trademarks of the lipid-induced
toxicity observed in pancreatic β-cells (Pineau et al., 2009) (Figure1A). When grown in the absence of
δ-ALA, this strain fails to synthesize haem, the prosthetic group of Ole1p, the fatty acid desaturase.
As a consequence, the hem1Δ cells stop growing as early as 5 hours after shift to non-δ-ALA
supplemented medium (YPD + Erg; Ø) and this arrest correlates with an accumulation of SFA (mainly
myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids) at the expense of unsaturated forms
(UFAs; palmitoleic (C16:1) and oleic (C18:1) acids) (Ferreira et al., 2004; Pineau et al., 2008).
Interestingly, cell growth can be fully recovered and all the trademarks of SFA-lipointoxication
alleviated if selective UFAs, such as the mono-unsaturated fatty acid oleic acid (Ole), are added to
the medium ((Deguil et al., 2011); Table S1 and Figure 1A).
However, Ole presents an important limitation as a potential curing agent. Indeed, the inability to
incorporate excess exogenous Ole into triacylglycerols (TAG) in a yeast mutant lacking the
to as QM (quadruple mutant) in the following sections), results in dysregulation of lipid synthesis,
massive proliferation of intracellular membranes, and ultimately cell death, in a process that appears
largely independent of the UPR pathway ((Petschnigg et al., 2009); Figure 1A). Importantly, the
same observations can also be reproduced in mammalian models (Listenberger et al., 2003). This
probably explains why Ole becomes toxic under lipointoxicated states, when the overall capacity to
buffer excess fatty acids within TAG pools is exceeded or, under normal conditions, to cells with low
TAG-synthesis capacity, such as islet non-beta cells (Cnop et al., 2001).
Journal of Cell Science • Advance article
acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis (that will be referred
The second step of our screening strategy therefore consisted in evaluating the innocuousness of
the molecules identified with the first screen (i. e. for their ability to restore the growth of hem1Δ
cells under SFA accumulation) towards the QM strain.
Based on our previous work, 64 fatty acids of various chain length and levels of unsaturation
were first tested (Table S1). Interestingly, in contrast to Ole, among the 12 UFAs which had proven to
be efficient in countering SFA-toxicity in the hem1Δ model (Deguil et al., 2011), 9 appeared to be less
toxic to the QM than Ole (Table S1). However, it appeared that low toxicity systematically correlated
with a lower efficiency to counter SFA-deleterious effects on growth (compare Ole and Cis vaccenic
acid, for example). Therefore, none of the fatty acids identified in this test proved to be as efficient
as Ole in relieving SFA-intoxication.
These results led us to initiate a broader screening process and to apply this two-step yeastbased assay to commercial molecule libraries (see the Materials and Methods section). Among the
1470 molecules tested in this study, only two compounds appeared to counter SFA toxicity as
efficiently as Ole in the hem1Δ model, with low toxicity to the quadruple mutant strain and the wildtype strain (Figure 1B-F).
Both of them, namely 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1-oleoyl lysophosphatidic acid
(OLPA) shared very similar structures (Figure 1C), with an oleic acyl chain at the sn-1 position.
Consistent with the lack of toxicity in the quadruple mutant strain, and in contrast to Ole, OLPA and
(Figure S1).
OLPA and OAG relieve the ER-stress in yeast and beta cells
Since it has been shown that the essential features required to promote viability reflects the
ability of fatty acids to alleviate ER stress both in yeast and mammalian cells (Deguil et al., 2011;
Dhayal and Morgan, 2011; Pineau et al., 2009), the capacity of OAG and OLPA to counteract the SFArelated UPR-induction was tested (Figure 2). In yeast, induction of the UPR pathway was determined
through the activity of the β-galactosidase reporter gene, set under the control of 4 UPR Elements
Journal of Cell Science • Advance article
OAG did not induce the accumulation of triacylglycerol in a normal, TAG-synthesis competent strain
(UPREs; (Cox and Walter, 1996)). According to our expectations, OAG and OLPA relieved the SFAinduced ER-stress to an extent very similar to Ole (Figure 2A).
OAG and OLPA also potently protected BRIN-BD11 cells (a rat hybrid pancreatic β-cell line)
against palmitate-induced cytotoxicity (Figure 2B), and this in a way very similar to what had already
been reported for Ole (Dhayal and Morgan, 2011). Modulation of the UPR pathway was evaluated in
this cell line by monitoring the phosphorylation of eIF2α, since this represents an appropriate
surrogate for induction of ER stress via the protein kinase RNA-like endoplasmic reticulum kinase
(PERK)-dependent pathway in fatty acid-treated β-cells ((Dhayal and Morgan, 2011); Figure 2C).
Culture of BRIN-BD11 cells with palmitate resulted in increased phosphorylation of eIF2α, consistent
with the induction of ER stress. Both OAG and OLPA markedly reduced the extent of eIF2α
phosphorylation observed in cells cultured with palmitate (Figure 2C).
Overall, these data validate our yeast-based screening assay as an efficient tool to identify new
lipointoxication inhibitors with potent effects in mammalian cells.
Pharmacogenomics and lipidomics suggest different modes of action for Ole, OLPA and
OAG
Since both OLPA and OAG bear an oleic acyl chain at the sn-1 position, a possible explanation for
their suppressive effect could be that they directly act as Ole donors. To test this hypothesis, we
used the property of Saccharomyces cerevisiae cells to grow on a variety of carbon sources, including
deprived of glucose (YP medium) and the ability of Ole, OLPA or OAG to support growth under these
restrictive conditions was evaluated. As shown in Figure 3A, if Ole was very efficient in sustaining
hem1Δ growth, neither OLPA nor OAG proved to be as potent as Ole. However, by contrast to OAG,
some residual growth could be observed with OLPA (Figure 3A). Therefore, in terms of fatty acid
supply, the three lipid species followed the order: Ole>>OLPA>OAG.
In order to identify potential common pathways for Ole, OLPA and OAG actions on SFA-induced
cytotoxicity, we then performed a genomic analysis on hem1Δ cells by comparing their
Journal of Cell Science • Advance article
fatty acids (Hiltunen et al., 2003). Therefore, hem1Δ cells were grown on a complete medium
transcriptomic profiles under growth in minimum medium supplemented with Erg and Ole, OLPA, or
OAG and with Erg alone (SFA accumulation; Ø). Differential expression analysis, conducted with a
cut-off of 2 for fold-change (FC) and of 0.0025 for the P-value, identified a total of 46 genes with
changes in expression levels after Ole addition as compared to Ø representing the SFA-accumulation
(Figure 3B and Table S2). Among this list, 14 genes were directly related to sterol and fatty
acid/phospholipid metabolism (Table S2). OLPA addition resulted in the dysregulation of 17 genes,
13 of them being common to Ole. This overlap between Ole and OLPA suggested that the regulation
of common metabolic pathway could be shared by the two molecules to account for resistance to
SFA. A prime example was the induction of FAA1 (Table S2), which encodes a long chain fatty acylCoA synthetase displaying a strong preference for C18:1 fatty acids (Knoll et al., 1994). By contrast,
the addition of OAG to the culture was not associated with any changes in gene expression as
compared to the SFA-accumulating condition (Figure 3B).
Other elements in favor of different modes of action for OLPA and OAG came from lipidomic
approaches. Figure 3C shows the relative distribution of phosphatidylcholine (PC) species under the
various culture conditions, obtained by comparison of positive ion mode spectra. Growth in YPD +
Erg medium (SFA-accumulation; Ø) resulted in decreased amounts of PC species bearing long
unsaturated chains (36:2, 36:1, 34:2, 34:1, 32:2, 32:1) in favor of species with saturated acyl chains
(32:0, 30:0, 28:0, 26:0, 24:0), as compared to normal growth conditions (δ-ALA) (Figure 3C and
phospholipid species (Figure S3). Addition of Ole resulted in enrichment in the diunsaturated (DB=2)
36:2 species (Dioleoyl PC, DOPC), which corresponds to a PC bearing two Ole (18:1) chains (Figure 3C
and 3E; Figure S2A). A similar C18:1 enrichment within PC was also observed when the cells were
incubated with OLPA, showing that this molecule can act as a potent oleic chain donor and acceptor
(Figure 3C and Figure S2A). By contrast, OAG supplementation resulted in an increase of the
monounsaturated species PC 34:1 (Palmitoyl Oleoyl PC or POPC; Figure 3C and 3E; Figure S2A and
Figure S3). As a consequence, the saturation rate of PC remained remarkably high under OAG
Journal of Cell Science • Advance article
Figure S2A). Such rearrangements of the fatty acyl chain content were also observed in other
treatment (50%; Figure 3D). Since a 40% saturation ratio is enough to preclude yeast cell growth
(Pineau et al., 2008), we concluded from these experiments that, by contrast to OLPA, OAG curing
effects cannot be accounted for by a simple restoration of the PL content. However, the enrichment
of 34:1 lipid species revealed that some of the OAG Ole chains are hydrolyzed and redistributed
within phospholipids to form monounsaturated (DB=1) species (Figures 3C-E; Figure S2A and Figure
S3). Therefore, in terms of C18:1 distribution, the following order was observed: Ole>OLPA>OAG,
with Ole and OLPA preferentially forming DB=2 and OAG, DB=1 PL species.
OLPA and OAG counter SFA impacts on the late secretory pathway by restoring surface
voids
As mentioned above, the accumulation of SFA within PL also alters the late secretory pathway: it
precludes the delivery of a model plasma membrane transporter, the uracil permease Fur4p, to its
final destination, in a process that correlates with a reduction of vesicular budding efficiency at the
TGN (Payet et al., 2013). Saturated PL appear to do so by increasing lipid order within intracellular
membranes and therefore reducing surface voids which, in turn, alters the recruitment of Gcs1p, a
void-sensing protein of the Arf-Gap family required for the cycling of nascent vesicles emerging from
the trans-Golgi (Payet et al., 2013). Both OLPA and OAG efficiently restored Fur4p delivery to the
plasma membrane (Figure 4A) and Gcs1p recruitment to the TGN (Figure 4B).
Gcs1p binds to bilayers thanks to its Amphipatic Lipid Packing Sensor (ALPS), which recognizes
hydrophobic residues into large surface clefts that are formed in the bilayer (Vanni et al., 2013). Such
voids can be obtained by introducing conical lipids within flat bilayers (Vamparys et al., 2013). In line
with this hypothesis, we demonstrated that Gcs1p cannot bind to liposomes obtained with PC
species purified from hem1Δ cells grown under SFA-accumulation, because of increased lipid packing
related to high saturated PL amounts (Payet et al., 2013). This process could therefore account for
Gcs1p mislocalization in vivo ((Payet et al., 2013) and Figure 4B) and is likely to participate in the
observed reduction in vesicular budding from the TGN. Since OAG failed to restore the minimal
Journal of Cell Science • Advance article
surface voids by the following mechanism: peptide partitioning is driven by the insertion of
diunsaturated PL ratio required for Gcs1p recruitment, we therefore evaluated whether this
molecule could generate surface voids by itself when incorporated within a packed/highly ordered
membrane. Relevant to this hypothesis, OAG could be detected as a free form in hem1Δ cells when
supplemented to the medium at a mol% ratio of 1.6 ± 0.4% OAG/total PL and 5.7 ± 0.6% OAG/PC
(Figure S2B).
To address the effect of OAG on the activity of an ALPS-containing protein, we performed GAP
assay using Gcs1p and liposomes (Figure 4C). As previously shown (Antonny et al., 1997a; Bigay et
al., 2005), the GAP activity of Gcs1p was very low in case of Golgi-mix liposomes containing mainly
16:0-C18:1, monounsaturated phospholipids (see Materials and Methods). This mix was chosen
because 16:0-C18:1 PC was the most prominent species under OAG supplementation (Figure 3C-E;
Figures S2A and S3). This result demonstrated that the formation of monounsaturated species
related to partial OAG metabolization is not sufficient to account for the Gcs1p recruitment to the
TGN observed in vivo. By contrast, GAP activity of Gcs1p was increased 15-fold by the addition of the
conical lipid Dioleoyl Glycerol (DOG; Figure 4C), which creates lipid packing defects/surface voids
(Vamparys et al., 2013; Vanni et al., 2014; Vanni et al., 2013). As shown in Figure 4C, the addition of
OAG also increased the activity of Gcs1p. A significant induction of Gcs1p activity could be observed
in vitro with as low as 7 mol% OAG, a mol ratio that falls in the same order of magnitude than the
ratio measured in vivo (see above). However, a dose-response curve showed that the effect of
confirmed that OAG can create surface voids, albeit less efficiently than DOG.
To test further the hypothesis of OAG acting as a potent “nanospacer”, molecular dynamics (MD)
simulations were performed on bilayers composed either of a disaturated (DMPC) or a
monounsaturated PC species (POPC), in the presence or in the absence of OAG (Figure 5). Such
compositions were chosen because disaturated and monounsaturated PC forms were the
predominant species in yeast under OAG supplementation (Figure 3E). For comparison, a pure DOPC
bilayer, mimicking the situation encountered under Ole- and OLPA-supplementation conditions
Journal of Cell Science • Advance article
33 mol% of OAG was comparable to the effect of 13 mol% of DOG. Collectively, these results
(Figure 3E), was also simulated. Moreover, additional simulations were also performed with DOG to
compare its impacts relative to OAG on POPC bilayers (Vamparys et al., 2013). To evaluate the size
and probability of the surface voids under these different conditions, we used a recently developed
method of membrane surface analysis (Vamparys et al., 2013). This method allows the identification
of chemical defects, where hydrocarbon chains are accessible to the solvent, and geometrical
defects, i.e., voids deeper than the glycerol backbone.
The main results of these simulations are presented in Figure 5. Panels A and B show side and top
views of a snapshot for a POPC 67/OAG 33 (mol%) bilayer.
The results in terms of surface voids are presented in Figures 5C and 5D. These figures show that
incorporating 10% of OAG has a slight but significant effect both on DMPC and POPC, for which we
observed an increase of the size of surface voids, as monitored by the packing defect constant (pdc),
of 0.7 and 0.5 Å2, respectively. Of importance, pdc increased in a synergistic manner when saturated
acyl chains were replaced by monosaturated ones and when OAG or DOG were introduced into the
bilayer; the pdc followed the order: DMPC < DMPC 90/OAG 10 < POPC < POPC 90/OAG 10 ≤ POPC
67/OAG 33 < POPC 87/DOG 13 < DOPC. These simulation results nicely fit our experimental data.
First, OAG impacts on DMPC and POPC bilayers can fully account for the efficiency of this molecule
to improve Gcs1p recruitment to the TGN under SFA-accumulation in vivo (Figure 4B) and to
monounsaturated liposomes in vitro (Figure 4C). Second, the fact that POPC/OAG combinations fail
molecule to promote Gcs1p recruitment to the TGN, as compared to Ole and OLPA (Figure 4B).
Finally, these simulations also confirmed that OAG, with its single monounsaturated aliphatic tail, is
less efficient than DOG for promoting surface voids, as observed in vitro (Figure 4C).
In summary, these simulations suggest that OLPA and OAG can induce surface voids compatible
with Gcs1p adsorption, albeit according to different mechanisms. Whereas OAG likely acts as a
nanospacer, OLPA preferentially generates voids at the membrane surface by favoring the formation
of DOPC species (Figure 5E).
Journal of Cell Science • Advance article
to induce surface voids as efficiently as DOPC can also account for the lower capacity of this
Pharmacophore design for virtual screening: molecular shape does matter to counter
SFA-lipointoxication
Since OAG and OLPA share very similar structures, their common molecular features were used
to generate a pharmacophore for the in silico screening of 6.5 million commercially-available
molecules (Figure 6; see Materials and Methods for details). Using this virtual screen, 23 different
compounds were identified and were individually assayed for their ability to counter SFAlipointoxication in the hem1Δ model, and for their innocuousness towards the QM strain. Among
these 23 candidates, 6 proved to efficiently counter lipointoxication without significant impacts on
the QM growth.
Interestingly, this virtual screening revealed a strict requirement for the presence of an Ole chain
within the molecular structure, since all the non-Ole containing molecules which matched, at least
partly, the pharmacophore, failed to counter SFA-lipointoxication in our yeast model (Figure 6 and
Table S3). Lipidomic analyses allowed separating the 6 positive hits into two different categories, i. e.
OLPA-like molecules, leading to the accumulation of DOPC, and OAG-like candidates, which failed to
restore normal diunsaturated PL amounts (see the example of CM22 on Figure 6 and Figure S3).
Interestingly, the hits tested in this study proved to potently protect BRIN-BD11 cells against
palmitate-induced cytotoxicity (Figure S4).
CM22 appeared as a very interesting candidate, since it behaved as a fully non-hydrolizable
SFA-toxicity. These observations reinforced the idea that restoring the PL content is not an absolute
prerequisite to counter SFA impacts: as an alternative suppressive mechanism, OAG-related
candidates likely act as nanospacers to restore the surface voids compatible with cell
survival/growth restoration under SFA-lipointoxication.
Journal of Cell Science • Advance article
analogue of OAG (Figure S3: compare CM22 and ø), but still counteracted all the traits associated to
Discussion
The two-step screening assay used in this study led us to identify, among 1470 molecules of
various structures, two lipid compounds, namely OLPA and OAG, sharing very similar structural
features. These two molecules appear to counter all the trademarks of SFA-lipointoxication: they
relieve the ER stress (Figure 2) and restore normal protein trafficking in the late secretory pathway
(Figure 4).
A very important conclusion of the present study is that both molecules restore surface voids
within cellular membranes, albeit according to different mechanisms, as monitored by the
recruitment of the void-sensing protein Gcs1p to the TGN in vivo (Figure 4B) and to liposomes in
vitro (Figure 4C).
OLPA treatment correlates with the formation of the diunsaturated PL species such as DOPC
(Figure 3C and Figure S2). This is not a surprising observation, since OLPA is a central molecule both
in the remodeling and de novo synthesis of PL, as a substrate for acyltransferases and transacylases
(Yamashita et al., 2014). In this context, OLPA can be used both as an Ole donor and acceptor,
therefore resulting in the formation of the DOPC that bears two oleic acyl chains.
Diunsaturated PC such as DOPC, thanks to their two monounsaturated chains, generate more
packing defects and surface voids in the bilayer than the cylindrical disaturated PC DMPC ((Vamparys
liposomes made of DOPC, but also to liposomes elaborated with PC purified from hem1Δ cells grown
in the presence of δ-ALA ((Payet et al., 2013); 55% diunsaturated species, Figure 3E). By contrast,
Gcs1p recruitment was low to liposomes elaborated with DMPC or PC species isolated from hem1Δ
cells grown under SFA-accumulation ((Payet et al., 2013); 5% diunsaturated species, Figure 3E).
These observations are very consistent with the mechanism for membrane recruitment of Gcs1p in
yeast and its mammalian homologue ArfGAP1. These two proteins sense membrane curvature via
their common ALPS motif (Bigay et al., 2005), which inserts hydrophobic residues into large surface
Journal of Cell Science • Advance article
et al., 2013); Figure 5C and D). We have already demonstrated that Gcs1p can bind avidly to
voids/clefts into the bilayer (Vanni et al., 2014; Vanni et al., 2013). The data reported in this study
both in vivo (Figure 4B), in vitro (Figure 4C) and in silico (Figures 5C and 5D) suggest that OLPA
restores normal vesicular budding, and therefore plasma-membrane protein delivery to their final
destination, by favoring the production of diunsaturated PL species.
The hypersensitivity of Gcs1 and ArfGAP1 to lipid packing makes them acute reporters of changes
in the packing properties of cellular membranes, but this does not imply that these changes affect
only these proteins. In fact, any protein that uses hydrophobic membrane insertion should be
sensitive to the lipid unsaturation ratio. However, the degree of sensitivity depends on the balance
between different modes of membrane interaction. For example, the amphipathic protein synuclein is not only sensitive to lipid packing but also to the presence of anionic lipids due to its
highly charged character (Pranke et al., 2011), which complicates the analysis. This is why we
focused here on the ALPS protein Gcs1p.
One may consider that OLPA is also a bioactive lipid, which is known to be involved in several
physiological and physiopathological pathways. Relevant to lipointoxication, OLPA has been shown
to activate the Peroxisome Proliferator Activated Receptor-γ (PPARy) in mammalian cells (Stapleton
et al., 2011). PPARy is involved in adipocyte differentiation and fatty acid metabolism (Stapleton et
al., 2011). However, such a system is not present in yeast, which lacks both nuclear hormone and
LPAx receptors (McIntyre et al., 2003). Therefore, if we cannot exclude that OLPA could help at
species is likely a prominent suppressive mechanism.
By contrast, OAG failed to induce the production of diunsaturated PC species, which remained in
remarkably low amounts (Figure 3C and E). OAG is a permeable analogue of DOG. Pioneering work in
mammalian cells suggested that, unlike DOG, OAG may directly intercalate into the phospholipid
bilayer and regulate downstream pathways (i. e. activation of Protein Kinase C (PKC)) without
interaction with specific cell receptors (Kaibuchi et al., 1983). Kaibuchi et al. suggested that DOG,
when exogenously supplied, is ineffective in inducing PKC activation, due to the steric hindrance
Journal of Cell Science • Advance article
countering SFA-intoxication in mammalian cells via PPARy induction, production of diunsaturated
related to its two long fatty acyl chains, which may preclude its intercalation within the phospholipid
bilayer (Kaibuchi et al., 1983). These observations nicely fit our data. Indeed, DOG (supplied as part
of the Bioactive lipid library and identified by the virtual screening (CM26, Figure 6)) proved to be
inefficient in countering SFA-related toxicity suggesting that the membrane-permeable properties of
OAG are a prerequisite for its action. Once intercalated within the membrane, OAG behaves in a way
very similar to DOG, by generating surface voids compatible with the recruitment of Gcs1p (Figures 4
and 5).
As in the case of OLPA, we cannot exclude that OAG may also exerts its suppressive effects in
mammalian cells by the regulation of downstream signaling pathways, notably the PKC pathway.
However, since Pkc1p, the main PKC yeast isoform, has been shown to be completely independent
of DOG for its activation (Antonsson et al., 1994), such a mechanism could not explain the extreme
efficiency of OAG in counteracting SFA toxicity in this model organism. This hypothesis was
reinforced by the fact that the DOG/OAG homologue 12-O-Tetradecanoylphorbol-13-acetate (TPA),
a potent inducer of PKC (Niedel et al., 1983), which displays a saturated acyl chain instead of an Ole
and does not match the pharmacophore developed in this study, failed to counter SFAlipointoxication in our cellular model (our unpublished data).
Several bulk properties of membranes, including their resistance to stretching (tension) and to
bending (bending modulus), are important parameters for cell physiology and also depend on the
might not exclusively occur through the formation of surface voids. However, it is interesting to note
that polyunsaturated fatty acids, which are efficiently incorporated within PL but poorly restore
yeast growth and Fur4p trafficking to the plasma membrane ((Deguil et al., 2011); Table S1), make
lipid membranes very flexible but are less prone to create deep surface voids than monounsaturated
lipids (Pinot et al., 2014; Rawicz et al., 2000). Therefore, the capacity to form surface voids seems at
roots of the effects reported here.
Journal of Cell Science • Advance article
level of lipid unsaturation (Rawicz et al., 2000). Therefore, the effects of C18 :1 containing molecules
To conclude, the present data point at modulation of membrane surface voids as a central
parameter to consider in order to efficiently counter the impacts of SFA on cell function in
lipointoxication-related human diseases. To our knowledge, this study is the first evidence that
targeting ubiquitous membrane biophysical properties with highly specific exogenously supplied
molecules could be of therapeutic relevance.
Materials and Methods
Yeast strains, mammalian cell line, culture conditions and reagents
Saccharomyces cerevisiae strains used in this study are listed in Table S2. The hem1Δ cells were
grown aerobically at 28 °C in YPD medium (1% yeast extract (w/v), 1% peptone (w/v), 2% Glucose
(w/v)) supplemented with 80 µg.mL-1 δ-aminolevulinate (δ-ALA). Accumulation of endogenous
saturated fatty acids (SFA) was obtained as previously described (Pineau et al., 2009) by inoculating
2.106 cells/mL, previously grown in YPD + δ-ALA, in YPD supplemented with 80 µg.mL-1 Ergosterol
(ERG; referred to as Ø), or alternatively, by seeding 3500 cells on the equivalent solid medium
containing 2% (w/v) agar. Compounds — such as oleic acid (Ole), 1-oleoyl-2-acetyl-sn-glycerol (OAG;
Cayman, Bertin Pharma, Montigny le Bretonneux, France) or 1-oleoyl-lysophosphatidic acid (OLPA;
Santa Cruz Biotechnology, Dallas, USA) — were supplemented, after cell transfer into/onto YPD + Erg
(Ø) liquid/solid medium, to assay their anti-SFA properties.
For Fur4p biogenesis studies, cells were transformed with the plasmid pFl38-Fur4p-green fluorescent
control of the inducible GAL10 promoter (Marchal et al., 2002). Localization of Gcs1p was conducted
using a single-copy plasmid expressing Gcs1p tagged at the amino terminus with GFP (pRS315-GFPGCS1; (Robinson et al., 2006)). Most effects of SFA accumulation were studied 7h after transfer to
YPD + ERG (Ø).
All products and reagents were purchased from Sigma-Aldrich, excepted where mentioned.
Journal of Cell Science • Advance article
protein (GFP), bearing a FUR4 fusion gene, encoding Fur4p with a C-terminal GFP tag under the
Compound screening
For the screening process, the Prestwick Chemical Library® (Prestwick, IllKirch, France), which
contains 1280 small molecules, 100% approved drugs, and the Screen-Well® Bioactive lipid library
(Enzo® Life Science, Farmingdale, USA), consisting of 190 lipid compounds of various structures,
were used.
To evaluate the anti-SFA propensities of these molecules, hem1Δ were seeded on the surface of YPD
+ Erg (Ø) plates as described above, and 5 µL drops of 1, 10 or 100 mM stock solutions (diluted in
dimethyl sulfoxide (DMSO) or ethanol (EtOH) solvents) were spotted prior to incubation at 28°C for 3
days. Colony formation at a specific spot reflects the ability of the corresponding compound to
rescue SFA-blocked hem1∆ proliferation (see (Deguil et al., 2011)). To evaluate the effects of the
candidate molecules in liquid cultures, the compounds were added at a 200 µM final concentration
to YPD + ERG medium. Proliferation kinetics were followed by measuring the optical density of the
culture at 600 nm (OD600 nm) every single hour (1 OD600 nm unit corresponding to 2.107 cells/mL).
In parallel, the innocuousness of the various compounds to a strain unable to store excess fatty acids
within lipid droplets was also evaluated. In this aim, WT and QM strains were grown in YPD medium,
under gentle shaking at 28°C and in aerobic conditions, prior to seeding 3500 respective cells/cm 2
onto YPD + agar 2% (w/v) plates. 1 µL drops of 1, 10 and 100 mM compound solutions (into EtOH or
inhibition halos were used as indicators of compound toxicity.
Lipid extraction, phospholipid purification and mass spectrometry (MS) analyses
hem1∆ (inoculated at a 2.107 cells/mL concentration) were grown in YPD + δ-ALA or YPD + Erg,
supplemented or not with 200 µM of the candidate molecules, under gentle shaking for 7 h, at 28°C
and in aerobic conditions. Quantitation of the various lipid species was performed by the mean of
internal standards (PC, PE, PI, PS and PA 25:0, http://avantilipids.com/), which were added before
extraction. Phospholipids were extracted and purified from 108 cells as already described (Payet et
Journal of Cell Science • Advance article
DMSO), were spotted onto agar prior to incubation at 28°C for 3 days. The diameters of growth
al., 2013). Purified PL were evaporated and reconstituted in 100 µL 2-propanol/methanol/water
(2:1:1, v:v:v) with 0.1% (v/v) formic acid for positive mode (PC analysis) and in 100 µL 2propanol/methanol/water (2:1:1, v:v:v) with 0.1% (v/v) triethylamine for negative mode (PA, PE, PI,
PS analysis).
The MS analysis was performed on a Waters Synapt G2 HDMS with a nanoESI source operated in
negative and positive ionization modes. The scan range was from 150 to 1200 m/z. Data analysis was
performed by using the LipidXplorer software (https://wiki.mpi-cbg.de/lipidx/Main_Page).
Because OAG is a relatively nonpolar compound, its quantification was assayed by APCI
(Atmospheric-pressure chemical ionization)-LC-MS/MS rather than ESI-MS. In this aim, dried lipid
samples obtained as described above were dissolved with 250 µl of CHCl3/CH3OH/H2O 60/30/4.5
v/v/v and 2 µL were injected on a 1200 6460-QqQ LC-MS/MS system equipped with an APCI source
(Agilent technologies). Separation was achieved on a Poroshell C8 2.1x100 mm, 2.7 µm column
(Agilent technologies) and acquisition was performed in Atmospheric Pressure Chemical Ionization
(APCI) positive Single Ion Monitoring (SIM) mode. Calibration curve was obtained using known
quantities of authentic standard (0,4 up to 2,5 nmol on column). The amount of free OAG per 108
hem1∆ cells was determined after 7 hours of growth in YPD + Erg supplemented with 200 µM OAG
and extensive washing of the cells with water. Non-specific adsorption of OAG was evaluated in a
parallel determination performed on 108 hem1∆ cells incubated with OAG for 2 minutes. OAG
Determination of UPR induction in the yeast model
hem1∆ transformed with the plasmid pPW344 (2µ URA3 4×UPRE-LacZ; (Cox and Walter, 1996)) were
grown into YPD + δ-ALA or YPD + Erg, supplemented or not with 200 µM of the compound of
interest, under gentle shaking at 28°C and in aerobic conditions for 7h. Then, 10 8 cells were
harvested in order to quantify the beta-galactosidase (β-gal) activity resulting from the UPRtriggered LacZ transgene expression, as described elsewhere (Pineau et al., 2009).
Journal of Cell Science • Advance article
amounts were finally expressed as mol% ratio of OAG/total PL and OAG/PC.
SFA lipointoxication in the β-pancreatic rat cell line BRIN-BD11
Palmitic acid stock was prepared in 50% (v/v) ethanol by heating to 70°C and then bound to FA-free
BSA by incubation at 37°C for 1h.
BRIN-BD11 cells were cultured in RPMI-1640 medium containing 11 mM glucose and supplemented
with 10% (w/v) fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL
streptomycin. Cells were seeded at a density of 0.5.105 cells/mL in 6-well plates in complete medium
for 24h at the beginning of each experiment. Then, the medium was removed and replaced by a
serum free version containing appropriate reagents complexed with BSA. Relevant vehicle (BSA plus
ethanol) controls were included in each experiment. Cell viability under the various treatments was
determined by propidium iodide staining as already described (Dhayal and Morgan, 2011).
To evaluate UPR induction, BRIN-BD11 cells were seeded at a density of 5.105 cells/mL in T25 flasks
in complete medium for 24 h. The medium was then removed and replaced by a serum-free version
containing appropriate BSA-complexed compounds, for 6 h. After treatment, whole cell proteins
were extracted and the ratio of phospho-eIF2α over total eIF2α was determined as described
elsewhere (Dhayal and Morgan, 2011).
Microarrays
RNAs were extracted from 2.107 hem1Δ cells using a commercial kit (RNeasy Mini Kit, Qiagen). For
each growth condition, samples were prepared from 3 individual biological samples. Agilent’s Yeast
strain of Saccharomyces cerevisiae. The ORF list was downloaded from the Saccharomyces Genome
Database (SGD: http://www.yeastgenome.org/) at Stanford University. For statistical confidences
and determination of differential gene expression, data files were analyzed using the free software R
associated to packages of “Bioconductor“ project. Developed workflows begin by data normalization
using Robust Multichip Average Method (RMA) (Irizarry et al., 2003) followed by a data analysis
workflow consisting in selecting differentially expressed genes. The statistical algorithm SAM
(Significance Analysis of Microarrays) [SAM] (Tusher et al., 2001) was used to compute P-value and
Journal of Cell Science • Advance article
oligo microarray contains 60-mer oligonucleotide probes to 6,256 annotated ORFs for the S288C
fold change for each gene. Selected genes were obtained using a cut-off both for fold change and Pvalue (FC Cutoff=2, P-value Cutoff=0.0025).
Confocal microscopy
Samples were examined by confocal laser scanning microscopy using a Bio-Rad MRC 1024 equipped
with a 15-mW argon-krypton gas laser. BodiPY, GFP fusion proteins and Sec7p-RFP were visualized
by excitation at 488 nm and 568 nm respectively, and with a 522 nm pass band filter or a 605 nm
pass band filter respectively (35 nm width). Quantification of Fur4p-GFP at the plasma membrane
was performed according to the procedure described by Nalaskowski et al. (Nalaskowski et al.,
2011). For each experiment, a minimum of 60 cells were randomly selected and examined. To
determine the plasma membrane/intracellular ratio, GFP fluorescence intensities both over the
plasma membrane and in intracellular areas, in each cell, were averaged to calculate the ratio of
plasma membrane over intracellular intensity. For the GFP-Gcs1p (expressed from a single-copy
plasmid encoding the Gcs1p protein tagged at the amino terminus with GFP (pRS315-GFP-GCS1;
(Robinson et al., 2006))) and Sec7p-RFP co-localization assay, a four-step analysis was carried out.
First, from the microscopy images, only GFP-expressing cells were selected and retained. Then,
among these selected cells, RFP fluorescence-spots were defined. Finally, the GFP fluorescence
intensities were quantified into these specific areas. To determine the co-localization scores, the RFP
dot-specific GFP intensity and the global GFP intensity were averaged. For evaluation of data,
given.
Tryptophan fluorescence GAP assay
Liposome preparation and GAP assay were carried out as previously described (Bigay et al., 2005)
with some modifications. Briefly, all lipids in chloroform were purchased from Avanti Polar Lipid. The
standard composition of Golgi-mix liposomes was (mol%) 1-palmitoyl-2-oleoylphosphatidylcholine
(POPC)
(50),
1-palmitoyl-2-oleoylphosphatidylethanolamine
(POPE)
(19),
1-palmitoyl-2-
oleoylphosphatidylserine (POPS) (5), liver phosphatidylinositol (PI) (10), cholesterol (16). When
Journal of Cell Science • Advance article
unpaired Student's t test was used using the Graphpad Prism 5 software. Mean values and S.E.M are
indicated, OAG (7, 13, 23 or 33 mol%) or 1,2-dioleoylglycerol (DOG) (13 mol%) were further added to
the Golgi-mix lipids in chloroform. Tryptophan fluorescence at 37°C was measured at 340 nm
(bandwidth 20 nm) upon excitation at 298 nm (bandwidth 1 nm) using JASCO FP-8300 fluorimeter.
The fluorescence quartz cuvette contained initially the liposomes (200 µM) in HKM buffer (HK + 1
mM MgCl2, 1 mM DTT). Thereafter, Arf1-GDP (0.4 µM) at 60 sec, GTP (50 µM) at 120 sec, EDTA (2
mM) at 180 sec, MgCl2 (2 mM) at 780 sec, Gcs1p (50 nM) at 840 sec were added sequentially and the
fluorescence was monitored in real time. The fluorescence intensities at 840 sec were normalized to
100. The apparent half-time (t1/2) of Arf deactivation was determined graphically as previously
described (Antonny et al., 1997b).
Molecular dynamics simulations
Molecular dynamics (MD) were performed to understand the effect of adding OAG in terms of
packing defects (which promote the adhesion of peripheral proteins). Previous 200 ns simulations of
pure DMPC, pure POPC and pure DOPC (Vamparys et al., 2013) were extended to 400 ns in this
work. Additional simulations of DMPC90/OAG10, POPC/OAG10, POPC/OAG33 and POPC87/DOG13
(all in molar ratio) were performed. The composition DOPC85/DOG15 of Vamparys et al. (Vamparys
et al., 2013) was also taken for comparison, but it was not extended to 400 ns like for the other
compositions. Each lipid system consisted of a patch of 256 lipids hydrated with 40 to 43 water
molecules per lipid (according to the different lipid compositions).
(Berger et al., 1997) was used for DMPC, POPC and DOPC and we used our previous model for DOG
(González-Rubio et al., 2011; Vamparys et al., 2013; Vanni et al., 2013). The topology of OAG was
copied from DOG. The systems were simulated for 400 ns and the analysis was performed on the
last 300 ns (except for DOPC85/DOG15). Each simulation was analyzed in terms of packing defects as
done previously (Pinot et al., 2014; Vamparys et al., 2013; Vanni et al., 2014; Vanni et al., 2013).
Briefly, we used a grid (with a granularity of 1 Å) parallel to the plane of the bilayer. We scan each
grid point vertically starting from outside of the bilayer towards its interior (water is excluded from
Journal of Cell Science • Advance article
All MD simulations were performed with GROMACS 4.5.3 (Hess et al., 2008). The Berger force field
the analysis) up to 1 Å below the sn-2 carbon atom of the nearest glycerol. If only lipid aliphatic
atoms are encountered the grid point is considered as an elementary packing defect of 1 Å2; if any
lipid polar atom is encountered that grid point is not considered as a defect. Adjacent elementary
defects are then clustered using a connected component algorithm, and the area of each cluster is
calculated (a packing defect is thus a cluster of elementary defects). This analysis is done for each
leaflet separately. The obtained distributions are then fit to a mono-exponential decay: p(A) = b e-A/d,
where p(A) is the probability of finding a defect of area A Å2, d is the exponential decay in units of Å2
, and b a constant. We then define the packing defect constant pdc in units of Å2 as the inverse of
the decay (pdc = d-1). The higher this constant, the higher the probability of finding large defects. The
error bar reported for each pdc corresponds to the standard error of regression coefficient
estimated during the fitting procedure.
Virtual screening
The present study was carried out using software LigandScout (version 4.03). LigandScout is a
software tool that extract and interprets ligands and their macromolecular environment to create 3D
pharmacophore models (Wolber and Langer, 2005).
Starting from 2 well known molecules (1-oleoyl-2-acetyl-sn-glycerol, OAG and 1-oleoyllysophosphatidic acid, OLPA), the Ligand-Based Modeling method was used. A maximum of 200
conformations were generated for each molecule with OMEGA-best settings. Then, a set of 10
pharmacophore optimization, the pharmacophores were validated. A database of 80 fatty acid
derivatives containing 14 tested active and 66 inactive compounds on the desired target (Deguil et
al., 2011) was screened against these pharmacophores, without taking into account the exclusion
volumes. The quality of the pharmacophore models was quantitatively evaluated by calculating the
selectivity, specificity and accuracy for each model separately (Braga and Andrade, 2013). Virtual
screening was performed on the selected pharmacophore using LigandScout on different databases
Journal of Cell Science • Advance article
pharmacophores was generated, using the shared feature pharmacophore method. After
that represent around 6.5 million of commercially available compounds coming from the Zinc
database Drug-now subset and various commercial suppliers.
Statistical analysis
Except where mentioned, P values were calculated by a two-tailed t test, using the Graphpad Prism 5
software. Means ± SEM at least representative of triplicates are indicated, with n.s.: non-significant,
Journal of Cell Science • Advance article
***<0.001, **<0.01 and *<0.05.
Acknowledgements
We are very grateful to Anne Cantereau (Confocal and Electron Microscopy facilities, University of
Poitiers, Poitiers, France) for her precious advice regarding confocal microscopy imaging and Lidwine
Trouilh (Biochips Platform, LISBP, Toulouse, France) for performing microarray preparations and
analyses. We are also grateful to Dr. Jean-Paul Pais de Barros (Plateforme de Lipidomique, INSERM
UMR866, Dijon, France) for his help concerning OAG quantification and to Dr. Patrick Bazzini
(Prestwick Chemical, Illkirch, France) for the virtual screening reported in this study. This work was
supported by Diabetes UK (grant 12/0004505 to NGM), the French MENRT, the CNRS, the EFSD
(European Foundation for the Study of Diabetes; with a grant to MS and TF) and the FEDER (Fonds
Journal of Cell Science • Advance article
Européen de Développement Régional; with a grant to RFC and TF).
Journal of Cell Science • Advance article
Figures
Figure 1: Identification of two “non-toxic anti-SFA” compounds using a yeast-based twostep screening strategy.
(A) Schematic representation of the screening strategy. The left diagram illustrates the main
characteristics of the hem1∆ strain. When grown in the presence of δ-aminolevulinate (δ-ALA),
hem1∆ cells produce haem which serves as the prosthetic group for Ole1p, the unique Delta(9) fatty
acid desaturase in yeast. Therefore, under δ-ALA supplementation, an optimal equilibrium between
saturated fatty acids (SFAs) and their unsaturated forms (UFAs) can be obtained within membrane
phospholipids. However, when grown in the absence of δ-ALA (Ø), hem1∆ cells fail to convert their
endogenous SFAs into their unsaturated counterparts. As previously described (Pineau et al., 2009),
this notably favors a SFA-overload within membrane PL which leads to (i) a global increase of the
saturation ratio of PL, (ii) an increase in membrane order, (iii) the induction of the unfolded protein
response (UPR) and of an endoplasmic reticulum (ER) stress and, ultimately, (iv) growth arrest.
Moreover, our preceding work (Deguil et al., 2011) indicated that SFA deleterious effects can be
counteracted if exogenously supplied UFAs, such as oleic acid (exoOle), are added to the medium (Ø).
Indeed, Ole can be incorporated within PL and, as a consequence, can restore the biophysical
properties of the membranes to normal, with positive impacts on SFA-associated stresses. The right
diagram describes the quadruple mutant (QM) yeast strain used in this study, which is deleted for
the are1, are2, dga1 and lro1 genes. Because of this particularity, the QM is deficient in the synthesis
of neutral lipids which constitute the core of the lipid droplets (LD). In this context, exogenously
biosynthesis pathways. It has been demonstrated that the QM strain is especially sensitive to exoUFAs
and this has been correlated to an overproduction of UFA-rich PL, massive proliferation of the ER
membrane and, ultimately, cell death (Petschnigg et al., 2009). (B) This two-step screening strategy
was used to identify new “non-toxic anti-SFA” compounds/therapeutic candidates, that are,
molecules (i) able to restore SFA-blocked hem1∆ growth, (ii) without displaying significant toxicity
towards the QM strain. From a total of 1470 molecules tested, two ester-bounded oleoyl-glycerol
derivative hits were selected, namely OLPA and OAG. Their molecular structures are shown. (C)
Journal of Cell Science • Advance article
supplied FAs, if in excess, cannot be buffered within LD, but are preferentially channeled to PL
hem1Δ cells were pre-grown under control conditions (δ-ALA supplementation) to stationary phase
and submitted to SFA-lipointoxication as described in Materials and Methods. 5 µL drops of 1, 10 or
100 mM Ole, OLPA or OAG solutions were spotted onto agar prior to incubation at 28°C for 3 days,
as previously described (Deguil et al., 2011). Development of colonies at specific spot locations
reflects the ability of the compound to rescue SFA-blocked hem1∆ proliferation. QM (D) and WT (E)
cells were plated on control media and 1 µL drops of 1, 10 and 100 mM compound solutions were
spotted onto agar prior to incubation at 28°C for 3 days. The diameters of growth inhibition halos
were used as indicators of compound toxicity. (F) hem1Δ cells were pregrown under control
conditions to stationary phase and submitted to SFA-lipointoxication in liquid cultures in the absence
(Ø) or in the presence of δ-ALA, Ole, OLPA or OAG, and proliferation kinetics were followed over
Journal of Cell Science • Advance article
time, by determining the optical density (OD) of the culture at 600 nm.
Figure 2: OLPA and OAG relieve the UPR in yeast and mammalian β-cell and prevent SFArelated cell death.
elements (UPREs)) were grown under control conditions, or were submitted to SFA-lipointoxication
in the absence (YPD), or in the presence of 200 µM Ole, OLPA or OAG. After 7 h of growth, βgalactosidase (β-gal) activities (UPR induction) were quantified. (B) BRIN-BD11 β-cells were grown
for 18 h under control conditions (Ctrl) or under SFA-lipointoxication (250 µM palmitic acid (Palm)),
in the presence of increasing concentrations of either OLPA or OAG, as mentioned. At the end of this
incubation, both dead and live cells were harvested, submitted to propidium iodide (IP) staining and
the proportions of dead cells were quantified. (C) BRIN-BD11 were grown as previously, without (Ø)
Journal of Cell Science • Advance article
(A) hem1Δ [pPW344] cells (bearing the LacZ gene under the control of a promoter containing 4×UPR
or with 100 µM OLPA or OAG supplementations. Then, the whole cell proteins were extracted and
submitted to Western blot in order to quantify phosphorylated (P-) and total eIF2α levels.
Phosphorylated (P-) over total eIF2α ratios, which reflect a UPR induction, are displayed. Values are
Journal of Cell Science • Advance article
means ± SD representative of three independent experiments. A. U.: arbitrary units.
Figure 3: OLPA and OAG impacts on the transcriptome and the lipidome
(A) hem1Δ cells were grown on rich medium deprived of glucose (YP medium) and 5 µL drops of
10 mM Ole, OLPA or OAG solutions were spotted onto agar prior to incubation at 28°C for 3 days.
The development of colonies indicates the ability of the spotted molecule to serve as a potent fatty
cultures in the absence or in the presence 200 µM of Ole, OLPA or OAG, as described in Materials
and Methods. After 7 h of growth, cells were harvested and total RNAs were extracted and used for
differential gene expression analysis. Comparison of the resulting transcriptomic profiles led to the
characterization of 17 Ole-upregulated genes (yellow-encircled
genes (yellow-encircled
symbols), 29 Ole-downregulated
symbols), 4 OLPA-upregulated genes (red-encircled
downregulated genes (red-encircled
symbols), 13 OLPA-
symbols) but no OAG-dysregulated genes. 13 genes were
similarly sensitive to Ole and OLPA (see Table S2 for details). (C) hem1Δ were grown as previously,
Journal of Cell Science • Advance article
acid source to sustain yeast growth. (B) hem1Δ cells were grown under SFA-lipointoxication in liquid
total lipids were extracted and PL species were purified and analyzed by ESI-MS. Representative PC
profiles from positive ion mode analysis are shown. The total carbon chain length (x) and number of
carbon-carbon double bounds (y) of the main PC molecular species (x:y) are indicated. (D) The data
displayed in (C) and Figure S2 were used to quantify the ratio of saturated versus unsaturated acyl
chains within PC species (saturation index). To obtain this ratio, the percentages of saturated,
monounsaturated and diunsaturated species are multiplied by 1, 0.5 and 0, respectively. (E) Relative
percentage of saturated (DB (double bond)=0) versus monounsaturated (DB=1) and diunsaturated
Journal of Cell Science • Advance article
(DB=2) species.
Journal of Cell Science • Advance article
Figure 4: OLPA and OAG counter SFA impacts on the late secretory pathway.
(A) hem1Δ[pFl38-Fur4p-GFP] yeast cells were grown under control conditions (δ-ALA
supplementation), or were submitted to SFA-lipotoxication, in the absence (Ø), or in the presence of
200 µM Ole, OLPA or OAG. After 7 h of growth, production of the Fur4p-GFP was induced and the
fusion protein observed by confocal microscopy (upper images). Quantification of Fur4p plasmamembrane localization was achieved as described in Materials and Methods (lower diagram). (B)
After 7 h of growth in the mentioned conditions, hem1Δ SEC7-RFP yeast cells transformed with the
pRS315-GFP-GCS1 plasmid were used for visualization of the GFP-Gcs1p and Sec7p-RFP fusion
proteins, by confocal microscopy (upper images). Co-localization of the two proteins of interest was
quantified as described in Materials and Methods (lower diagram). A. U.: arbitrary units. (C)
Tryptophan fluorescence GAP assay. Liposomes (mol ratio) were prepared from a Golgi-mix (POGolgi) (black, 100), PO-Golgi/OAG (from light to dark blue: 93/7, 87/13, 77/23 and 67/33), POGolgi/DOG (red, 87/13). Arf1-GDP (0.4 µM) at 60 sec, GTP (50 µM) at 120 sec, EDTA (2 mM) at 180
sec, MgCl2 (2 mM) at 780 sec and Gcs1p (50 nM) at 840 sec were added sequentially. The
fluorescence intensities at 840 sec were normalized to 100. The inset shows the reciprocal of
apparent half-time (t1/2) as a function of the OAG mol ratio. The fluorescence data are one of two
Journal of Cell Science • Advance article
independent experiments. The values in the inset are mean ± S.D. of two independent experiments.
(A) Side view of a snapshot of POPC67/OAG33. Water is shown in blue sticks, lipids are shown in
spheres. Aliphatic atoms of POPC and OAG are colored in yellow and orange respectively. Polar
atoms of POPC and OAG are colored in dark and light grey, respectively. (B) Top view of the same
snapshot as panel (A). The color code is the same except for surface voids which are represented as
clusters of ice blue spheres (each sphere is an elementary surface void of 1 Å2). (C) Distributions of
Journal of Cell Science • Advance article
Figure 5: Size distribution of surface voids in model membrane systems.
surface voids for various lipid compositions: pure DMPC, DMPC90/OAG10, pure POPC,
POPC90/OAG10, POPC67/OAG33, POPC87/DOG13 and pure DOPC (colored according to a rainbow
palette). The solid colored lines are the exponential fits to the packing defect/surface void
distributions. The solid and dashed lines show the area used for the fit (A > 15 Å2 and p(A) ≥ 10-4). (D)
Packing defect constants in units of Å2 for the different lipid compositions (same color code as Panel
C). In each bar the value of the constant is written as well as the percentage compared to the
maximum pdc (pure DOPC). (E) A schematic representation summarizing OLPA/Ole and OAG actions
on SFA-overloaded membranes. Whereas OLPA and Ole efficiently metabolize their UFA moiety
within membrane PL to generate diunsaturated species, OAG may preferentially intercalate within
saturated membranes at first glance to act as a nanospacer. Both mechanisms restore surface voids
compatible with amphipathic lipid-packing sensor (ALPS) motif-containing proteins —such as Gcs1p
Journal of Cell Science • Advance article
in yeast— recruitment, membrane vesiculation and protein trafficking along the secretory pathway.
Journal of Cell Science • Advance article
Figure 6: The virtual screening strategy used in this study.
A pharmacophore summarizing the main structural features of OAG and OLPA was used to screen
for 6.5 million commercially-available molecules. The 23 candidates obtained were tested in our
yeast model for their ability to counter SFA-lipointoxication and for their innocuousness towards the
QM strain. Among them, 6 hit molecules displaying the expected properties were classified either as
OLPA-like or OAG-like candidates, according to their lipidomic profiles (see examples in the lower
Journal of Cell Science • Advance article
diagrams).
References
Akazawa, Y., S. Cazanave, J.L. Mott, N. Elmi, S.F. Bronk, S. Kohno, M.R. Charlton, and G.J. Gores.
2010. Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and
hepatocyte lipoapoptosis. Journal of Hepatology. 52:586-593.
Alkhateeb, H., A. Chabowski, J.F.C. Glatz, J.F.P. Luiken, and A. Bonen. 2007. Two phases of palmitateinduced insulin resistance in skeletal muscle: impaired GLUT4 translocation is followed by a
reduced GLUT4 intrinsic activity. American Journal of Physiology - Endocrinology And
Metabolism. 293:E783-E793.
Antonny, B., I. Huber, S. Paris, M. Chabre, and D. Cassel. 1997a. Activation of ADP-ribosylation factor
1 GTPase-activating protein by phosphatidylcholine-derived diacylglycerols. J Biol Chem.
272:30848-30851.
Antonny, B., I. Huber, S. Paris, M. Chabre, and D. Cassel. 1997b. Activation of ADP-ribosylation Factor
1 GTPase-Activating Protein by Phosphatidylcholine-derived Diacylglycerols. Journal of
Biological Chemistry. 272:30848-30851.
Antonsson, B., S. Montessuit, L. Friedli, M.A. Payton, and G. Paravicini. 1994. Protein kinase C in
yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product. Journal of
Biological Chemistry. 269:16821-16828.
Berger, O., O. Edholm, and F. Jähnig. 1997. Molecular dynamics simulations of a fluid bilayer of
dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant
temperature. Biophysical Journal. 72:2002-2013.
Bigay, J., and B. Antonny. 2012. Curvature, lipid packing, and electrostatics of membrane organelles:
defining cellular territories in determining specificity. Developmental cell. 23:886-895.
Bigay, J., J.F. Casella, G. Drin, B. Mesmin, and B. Antonny. 2005. ArfGAP1 responds to membrane
curvature through the folding of a lipid packing sensor motif. Embo J. 24:2244-2253.
Braga, R.C., and C.H. Andrade. 2013. Assessing the Performance of 3D Pharmacophore Models in
Virtual Screening: How Good are They? Current Topics in Medicinal Chemistry. 13:11271138.
Cnop, M., J.C. Hannaert, A. Hoorens, D.L. Eizirik, and D.G. Pipeleers. 2001. Inverse Relationship
Between Cytotoxicity of Free Fatty Acids in Pancreatic Islet Cells and Cellular Triglyceride
Accumulation. Diabetes. 50:1771-1777.
Cox, J.S., and P. Walter. 1996. A Novel Mechanism for Regulating Activity of a Transcription Factor
That Controls the Unfolded Protein Response. Cell. 87:391-404.
de Kroon, A.I.P.M. 2007. Metabolism of phosphatidylcholine and its implications for lipid acyl chain
composition in Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA) - Molecular
and Cell Biology of Lipids. 1771:343-352.
Deguil, J., L. Pineau, E.C. Rowland Snyder, S. Dupont, L. Beney, A. Gil, G. Frapper, and T. Ferreira.
2011. Modulation of lipid-induced ER stress by fatty acid shape. Traffic. 12:349-362.
Journal of Cell Science • Advance article
Borradaile, N.M., X. Han, J.D. Harp, S.E. Gale, D.S. Ory, and J.E. Schaffer. 2006. Disruption of
endoplasmic reticulum structure and integrity in lipotoxic cell death. J. Lipid Res. 47:27262737.
Dhayal, S., and N.G. Morgan. 2011. Structure-activity relationships influencing lipid-induced changes
in eIF2alpha phosphorylation and cell viability in BRIN-BD11 cells. FEBS letters. 585:22432248.
Ferreira, T., M. Regnacq, P. Alimardani, C. Moreau-Vauzelle, and T. Berges. 2004. Lipid dynamics in
yeast under haem-induced unsaturated fatty acid and / or sterol depletion. Biochem J.
378:899-908.
Gillon, A.D., C.F. Latham, and E.A. Miller. 2012. Vesicle-mediated ER export of proteins and lipids.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1821:1040-1049.
González-Rubio, P., R. Gautier, C. Etchebest, and P.F.J. Fuchs. 2011. Amphipathic-Lipid-PackingSensor interactions with lipids assessed by atomistic molecular dynamics. Biochimica et
Biophysica Acta (BBA) - Biomembranes. 1808:2119-2127.
Guo, W., S. Wong, W. Xie, T. Lei, and Z. Luo. 2007. Palmitate modulates intracellular signaling,
induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat
primary preadipocytes. Vol. 293. E576-586.
Gwiazda, K.S., T.-L.B. Yang, Y. Lin, and J.D. Johnson. 2009. Effects of palmitate on ER and cytosolic
Ca2+ homeostasis in {beta}-cells. Vol. 296. E690-701.
Hess, B., C. Kutzner, D. van der Spoel, and E. Lindahl. 2008. GROMACS 4: Algorithms for Highly
Efficient, Load-Balanced, and Scalable Molecular Simulation. Journal of chemical theory and
computation. 4:435-447.
Hiltunen, J.K., A.M. Mursula, H. Rottensteiner, R.K. Wierenga, A.J. Kastaniotis, and A. Gurvitz. 2003.
The biochemistry of peroxisomal β-oxidation in the yeast Saccharomyces cerevisiae. FEMS
Microbiology Reviews. 27:35-64.
Irizarry, R.A., B. Hobbs, F. Collin, Y.D. Beazer-Barclay, K.J. Antonellis, U. Scherf, and T.P. Speed. 2003.
Exploration, normalization, and summaries of high density oligonucleotide array probe level
data. Biostatistics. 4:249-264.
Kaibuchi, K., Y. Takai, M. Sawamura, M. Hoshijima, T. Fujikura, and Y. Nishizuka. 1983. Synergistic
functions of protein phosphorylation and calcium mobilization in platelet activation. Journal
of Biological Chemistry. 258:6701-6704.
Laybutt, D.R., A.M. Preston, M.C. Akerfeldt, J.G. Kench, A.K. Busch, A.V. Biankin, and T.J. Biden. 2007.
Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes.
Diabetologia. 50:752-763.
Listenberger, L.L., X. Han, S.E. Lewis, S. Cases, R.V. Farese, D.S. Ory, and J.E. Schaffer. 2003.
Triglyceride accumulation protects against fatty acid-induced lipotoxicity. PNAS. 100:30773082.
Marchal, C., S. Dupre, and D. Urban-Grimal. 2002. Casein kinase I controls a late step in the endocytic
trafficking of yeast uracil permease. J Cell Sci. 115:217-226.
McIntyre, T.M., A.V. Pontsler, A.R. Silva, A. St. Hilaire, Y. Xu, J.C. Hinshaw, G.A. Zimmerman, K. Hama,
J. Aoki, H. Arai, and G.D. Prestwich. 2003. Identification of an intracellular receptor for
lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist. Proceedings of the National
Academy of Sciences. 100:131-136.
Journal of Cell Science • Advance article
Knoll, L.J., D.R. Johnson, and J.I. Gordon. 1994. Biochemical studies of three Saccharomyces
cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p. Journal of Biological Chemistry.
269:16348-16356.
Miller, T.A., N.K. LeBrasseur, G.M. Cote, M.P. Trucillo, D.R. Pimentel, Y. Ido, N.B. Ruderman, and D.B.
Sawyer. 2005. Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes.
Biochemical and Biophysical Research Communications. 336:309-315.
Nalaskowski, M.M., R. Fliegert, O. Ernst, M.A. Brehm, W. Fanick, S. Windhorst, H. Lin, S. Giehler, J.
Hein, Y.-N. Lin, and G.W. Mayr. 2011. Human Inositol 1,4,5-Trisphosphate 3-Kinase Isoform B
(IP3KB) Is a Nucleocytoplasmic Shuttling Protein Specifically Enriched at Cortical Actin
Filaments and at Invaginations of the Nuclear Envelope. Journal of Biological Chemistry.
286:4500-4510.
Niedel, J.E., L.J. Kuhn, and G.R. Vandenbark. 1983. Phorbol diester receptor copurifies with protein
kinase C. Proceedings of the National Academy of Sciences of the United States of America.
80:36-40.
Payet, L.A., L. Pineau, E.C. Snyder, J. Colas, A. Moussa, B. Vannier, J. Bigay, J. Clarhaut, F. Becq, J.M.
Berjeaud, C. Vandebrouck, and T. Ferreira. 2013. Saturated fatty acids alter the late
secretory pathway by modulating membrane properties. Traffic. 14:1228-1241.
Petschnigg, J., H. Wolinski, D. Kolb, G.n. Zellnig, C.F. Kurat, K. Natter, and S.D. Kohlwein. 2009. Good
Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane
Homeostasis in Yeast. J Biol Chem. 284:30981-30993.
Pineau, L., L. Bonifait, J.-M. Berjeaud, P. Alimardani-Theuil, T. Berges, and T. Ferreira. 2008. A Lipidmediated Quality Control Process in the Golgi Apparatus in Yeast. Mol. Biol. Cell. 19:807-821.
Pineau, L., J. Colas, S. Dupont, L. Beney, P. Fleurat-Lessard, J.M. Berjeaud, T. Berges, and T. Ferreira.
2009. Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids. Traffic.
10:673-690.
Pinot, M., S. Vanni, S. Pagnotta, S. Lacas-Gervais, L.A. Payet, T. Ferreira, R. Gautier, B. Goud, B.
Antonny, and H. Barelli. 2014. Lipid cell biology. Polyunsaturated phospholipids facilitate
membrane deformation and fission by endocytic proteins. Science. 345:693-697.
Pranke, I.M., V. Morello, J. Bigay, K. Gibson, J.M. Verbavatz, B. Antonny, and C.L. Jackson. 2011.
alpha-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting
chemistry mediates selective vesicle binding. J Cell Biol. 194:89-103.
Preston, A., E. Gurisik, C. Bartley, D. Laybutt, and T. Biden. 2009. Reduced endoplasmic reticulum
(ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by
promoting protein overload. Diabetologia. 52:2369-2373.
Robinson, M., P.P. Poon, C. Schindler, L.E. Murray, R. Kama, G. Gabriely, R.A. Singer, A. Spang, G.C.
Johnston, and J.E. Gerst. 2006. The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the
Golgi in yeast. Mol Biol Cell. 17:1845-1858.
Schneiter, R., B. Brugger, R. Sandhoff, G. Zellnig, A. Leber, M. Lampl, K. Athenstaedt, C. Hrastnik, S.
Eder, G. Daum, F. Paltauf, F.T. Wieland, and S.D. Kohlwein. 1999. Electrospray ionization
tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition
of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct
molecular species en route to the plasma membrane. J Cell Biol. 146:741-754.
Stapleton, C.M., D.G. Mashek, S. Wang, C.A. Nagle, G.W. Cline, P. Thuillier, L.M. Leesnitzer, L.O. Li,
J.B. Stimmel, G.I. Shulman, and R.A. Coleman. 2011. Lysophosphatidic Acid Activates
Peroxisome Proliferator Activated Receptor-γ in CHO Cells That Over-Express Glycerol 3Phosphate Acyltransferase-1. PLoS ONE. 6:e18932.
Journal of Cell Science • Advance article
Rawicz, W., K.C. Olbrich, T. McIntosh, D. Needham, and E. Evans. 2000. Effect of chain length and
unsaturation on elasticity of lipid bilayers. Biophys J. 79:328-339.
Tamura, Y., H. Sesaki, and T. Endo. 2014. Phospholipid Transport via Mitochondria. Traffic. 15:933945.
Tusher, V.G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the
ionizing radiation response. Proceedings of the National Academy of Sciences of the United
States of America. 98:5116-5121.
Vamparys, L., R. Gautier, S. Vanni, W.F.D. Bennett, D.P. Tieleman, B. Antonny, C. Etchebest, and
Patrick F.J. Fuchs. 2013. Conical Lipids in Flat Bilayers Induce Packing Defects Similar to that
Induced by Positive Curvature. Biophysical Journal. 104:585-593.
Vanni, S., H. Hirose, H. Barelli, B. Antonny, and R. Gautier. 2014. A sub-nanometre view of how
membrane curvature and composition modulate lipid packing and protein recruitment. Nat
Commun. 5:4916.
Vanni, S., L. Vamparys, R. Gautier, G. Drin, C. Etchebest, Patrick F.J. Fuchs, and B. Antonny. 2013.
Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic
Residues. Biophysical Journal. 104:575-584.
Wolber, G., and T. Langer. 2005. LigandScout: 3-D Pharmacophores Derived from Protein-Bound
Ligands and Their Use as Virtual Screening Filters. Journal of chemical information and
modeling. 45:160-169.
Journal of Cell Science • Advance article
Yamashita, A., Y. Hayashi, Y. Nemoto-Sasaki, M. Ito, S. Oka, T. Tanikawa, K. Waku, and T. Sugiura.
2014. Acyltransferases and transacylases that determine the fatty acid composition of
glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model
organisms. Progress in Lipid Research. 53:18-81.
Figure S1: Impacts of OLPA and OAG supplementation of Lipid Droplet (LD) formation.
hem1Δ cells were grown under control conditions (δ-ALA supplementation), or were submitted to
SFA-lipotoxication, in the absence (Ø), or in the presence of 200 µM Ole, OLPA or OAG. After 7 h of
growth, cells were loaded with 2 μM of the fluorescent probe BodiPY in order to observe lipid droplet
by confocal microscopy, as described in Materials and Methods.
Journal of Cell Science • Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
Figure S2: Phospholipid species under the different culture conditions.
(A)
hem1Δ were grown under control conditions (δ-ALA supplementation), or were
submitted to SFA-lipotoxication, in the absence (Ø), or in the presence of 200 µM Ole, OLPA
or OAG, before lipid extraction and ESI-MS analysis of PC species in the positive ion mode. The
relative abundance of each molecular PC species was determined as described in Materials and
Methods. Values are means ± S. D. of at least three independent determinations. (B) hem1Δ were
grown in the media indicated as described above and the various PL classes were quantified by ESI-
Journal of Cell Science • Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
MS as described in the Materials and Methods section, using 25:0 PL as internal standards. OAG was
quantified by APCI-MS (see Materials and Methods for details). Lipid class composition is expressed
in mol% of total lipids in the sample. Values are means ± S. D. of at least two independent
determinations. An interesting observation is that, under Ole supplementation, accumulation of
C18:1 within PC correlated with an increase in the PC amounts relative to other PL classes, and more
specifically PE. PE, due to the comparatively small cross-sectional area of the polar head-group,
displays an overall conical shape and therefore behaves as a non-bilayer lipid (Boumann et al., 2006).
This non-lamellar propensity is increased if saturated acyl chains are substituted by kinked,
monounsaturated chains such as C18:1 (Boumann et al., 2006). Therefore, one may hypothesize that,
upon C18:1 accumulation within the cell, yeast limit its redistribution towards non-lamellar forming
Journal of Cell Science • Supplementary information
species but rather favors its partitioning within more cylindrical PLs such as PC.
Figure S3: The relative distribution of saturated (DB=0), monounsaturated (DB=1) and diunsaturated (DB=2) species within the various phospholipid classes. The data displayed are from a
representative experiment (from three independent experiments).
Journal of Cell Science • Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
Figure S4: OAG-like candidates prevent SFA-related cell death in mammalian β-cell.
BRIN-BD11 β-cells were grown for 18 h under control conditions (Ctrl) or under SFA-lipointoxication
(250 µM plamitic acid (Palm)), in the presence of increasing concentrations of either CM10, CM22 or
CM30, as mentioned. At the end of this incubation, both dead and live cells were harvested,
submitted to propidium iodide (IP) staining and the proportions of dead cells were quantified.
Journal of Cell Science • Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
Table S1: The various fatty acids tested in this study and their relative toxicity to a strain defective in
Triglyceride synthesis.
List of the various fatty acids tested in this study, their relative propensities to restore SFA-blocked
hem1∆ growth (data from (Deguil et al., 2011)) and their relative toxicities towards the quadruple
mutant (QM) or its wild type counterpart (WT). The diameters of growth inhibition halos were
determined in each case and were used as indicators of compound toxicity. x: number of carbons of
the acyl chain/y: number of unsaturation(s).
Journal of Cell Science • Supplementary information
Click here to Download Table S1
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
Table S2: Full-list of the genes dysregulated by Ole or OLPA additions under SFA-lipotointoxication.
hem1Δ cells were grown under SFA-lipointoxication in the absence or in the presence 200 µM of Ole
or OLPA, as described in Experimental procedures. After 7 h of growth, cells were harvested and total
RNAs were extracted and used for differential gene expression analysis. Log 2 Fold Change (Log2FC)
and Adjusted (Adj.) P values are relative to SFA-lipointoxication conditions (Erg supplementation
alone).
Journal of Cell Science • Supplementary information
Click here to Download Table S2
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
Table S3: Structures of the main candidates identified by virtual screening.
Table S4: Characteristics of the yeast strains used in this study.
Click here to Download Table S4
Journal of Cell Science • Supplementary information
Click here to Download Table S3
J. Cell Sci. 129: doi:10.1242/jcs.183590: Supplementary information
References
Journal of Cell Science • Supplementary information
Boumann, H.A., J. Gubbens, M.C. Koorengevel, C.S. Oh, C.E. Martin, A.J. Heck, J. Patton-Vogt, S.A.
Henry, B. de Kruijff, and A.I. de Kroon. 2006. Depletion of phosphatidylcholine in yeast
induces shortening and increased saturation of the lipid acyl chains: evidence for regulation
of intrinsic membrane curvature in a eukaryote. Mol Biol Cell. 17:1006-1017.
Deguil, J., L. Pineau, E.C. Rowland Snyder, S. Dupont, L. Beney, A. Gil, G. Frapper, and T. Ferreira.
2011. Modulation of lipid-induced ER stress by fatty acid shape. Traffic. 12:349-362.