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COMMENTARY
Role of cholesterol in SNARE-mediated trafficking on intracellular
membranes
Carlos Enrich1,*, Carles Rentero1, Aitor Hierro2 and Thomas Grewal3,*
The cell surface delivery of extracellular matrix (ECM) and integrins
is fundamental for cell migration in wound healing and during cancer
cell metastasis. This process is not only driven by several soluble
NSF attachment protein (SNAP) receptor (SNARE) proteins,
which are key players in vesicle transport at the cell surface
and intracellular compartments, but is also tightly modulated by
cholesterol. Cholesterol-sensitive SNAREs at the cell surface are
relatively well characterized, but it is less well understood how
altered cholesterol levels in intracellular compartments impact on
SNARE localization and function. Recent insights from structural
biology, protein chemistry and cell microscopy have suggested
that a subset of the SNAREs engaged in exocytic and retrograde
pathways dynamically ‘sense’ cholesterol levels in the Golgi and
endosomal membranes. Hence, the transport routes that modulate
cellular cholesterol distribution appear to trigger not only a change in
the location and functioning of SNAREs at the cell surface but also
in endomembranes. In this Commentary, we will discuss how
disrupted cholesterol transport through the Golgi and endosomal
compartments ultimately controls SNARE-mediated delivery of
ECM and integrins to the cell surface and, consequently, cell
migration.
KEY WORDS: SNARE protein, Cholesterol, Trans-Golgi network,
Recycling endosome, Integrin trafficking, Cell migration
Introduction
Cholesterol homeostasis is essential for the functional integrity of
the cell and requires a tight regulation of cholesterol levels inside
and outside of cells. Therefore, cholesterol-sensing regulatory
circuits communicate any alterations in cholesterol levels in order
to control sterol synthesis, uptake and transport (Ikonen, 2008;
Maxfield and van Meer, 2010).
Cholesterol is considered to be indispensable for cell migration.
A substantial body of literature suggests that specialized
cholesterol-rich microdomains and cholesterol-sensitive transport
pathways are required for the delivery of integrins and extracellular
matrix (ECM) to and from the cell surface. Yet, despite the now
well-recognized increased cholesterol demand in cancer cells, it
remains unclear how intracellular cholesterol transport routes are
linked with the molecular machinery that controls cell migration.
1
Departament de Biologia Cellular, Immunologia i Neurociències, Centre de
Recerca Biomèdica CELLEX, Institut d’Investigacions Biomèdiques August Pi i
Sunyer (IDIBAPS). Facultat de Medicina, Universitat de Barcelona, 08036Barcelona, Spain. 2Structural Biology Unit, CIC bioGUNE, Bizkaia Technology
Park, 48160 Derio; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao,
Spain. 3Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.
*Authors for correspondence ([email protected]; [email protected])
Here, we will first summarize our current knowledge of
cholesterol homeostasis, in particular the cellular distribution of
low-density lipoprotein (LDL), which is linked to events that are
highly relevant for cell migration. We will then focus on how
cholesterol transport along intracellular routes can modulate the
localization of several NSF attachment protein (SNAP) receptor
(SNARE) proteins, major players in membrane transport to the
cell surface along endocytic and exocytic pathways. Finally we
will discuss how localization of cholesterol-sensitive SNAREs, in
particular within Golgi–endosomal boundaries, determines the
efficacy of delivery of cell adhesion receptors and ECM, which
are key factors in cancer cell migration, to the cell surface.
Cholesterol has multiple features that enable interaction
with lipids and proteins
Cholesterol is an amphipathic molecule with a hydrophilic
hydroxyl group and a hydrophobic section organized into four
rigid rings followed by a short branched hydrocarbon tail. Within
the membrane, the hydroxyl group of cholesterol interacts
with the polar head of phospholipids and sphingolipids,
whereas the hydrophobic part remains immersed in the
membrane alongside the hydrocarbon chains of the surrounding
lipids. Cholesterol is not uniformly distributed in the membrane,
and besides its interaction with membrane lipids, interaction with
proteins also have an important role in its distribution (Epand,
2006; Ikonen and Jansen, 2008). Consequently, there has been a
considerable interest on deciphering the molecular mechanisms
that allow these interactions. Indeed, several posttranslational
modifications appear to enable proteins to preferentially associate
with cholesterol-rich domains, including their myristoylation,
palmitoylation or glycosylphosphatidylinositol linkage. However,
for cholesterol to interact with transmembrane domains of
proteins, one would expect to find specific molecular features
at the lipid–water interface region of proteins such as defined
amino acid sequences or motifs. Indeed, several short cholesterolbinding motifs that are located in the lipid–water interface region
favor interactions with cholesterol (Box 1). In some cases,
the precise positioning of a cholesterol-binding motif within
the lipid–water interface segment might be influenced by the
‘snorkelling’ effect seen for lysine and arginine residues with
long buried hydrophobic side-chains (Chamberlain et al., 2004;
Strandberg and Killian, 2003). However, how such interactions
contribute to maintaining a heterogeneous distribution of
cholesterol within the cell and to cholesterol movement
between cellular membranes is not fully understood.
Intracellular trafficking of cholesterol
Several reviews have discussed the intracellular distribution of
cholesterol and cholesterol-rich membrane domains, pathways of
intracellular cholesterol movement and methods for studying
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Journal of Cell Science
ABSTRACT
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
Box 1. Cholesterol-binding motifs in proteins
The CRAC (cholesterol recognition/interaction amino acid
consensus) represents the best-characterized cholesterol-binding
motif (Fantini and Barrantes, 2013; Li and Papadopoulos, 1998) and
conforms to the pattern: L/V-X1–5-Y-X1–5-R/K (Fig. 2). Despite
some skepticism, CRAC is found in several cholesterol-binding
proteins, including caveolin-1 and the somatostatin receptor (Baier
et al., 2011; Murata et al., 1995). In addition, in many cases the
interaction between cholesterol and CRAC has been confirmed by
physicochemical or mutagenic analysis. An inverted CRAC
cholesterol-binding motif, the CARC domain [K/R-X1-5-Y/F-X1–5-L/
V], is found in some other proteins (Baier et al., 2011) (Fig. 2). In
addition, the transmembrane domains of several proteins (i.e. TM4
of the human b2-adrenergic receptor) contain a sequence with a
combination of basic (R), aromatic (W) and aliphatic (L/V) residues
(R-W-L, see Fig. 2B) that does not strictly fulfil the criteria of the
CARC algorithm, but still binds to cholesterol (Hanson et al., 2008).
Remarkably, the CRAC, CARC and R-W-L binding motifs exhibit a
common distribution of basic (K/R), aromatic (Y/F/W) and branched
aliphatic residues (L/V) that are important for cholesterol interaction.
Furthermore, in a-synuclein, the Alzheimer’s b-amyloid peptide and
the N-terminal peptide of HIV-1 gp41, the cholesterol-binding sites
are located in tilted peptides, which lack the basic and aromatic
residues characteristic of the previous motifs (Charloteaux et al.,
2006). The definition of tilted peptides is functional, not sequencebased, and collectively corresponds to short helical fragments
with an asymmetric distribution of their hydrophobic residues that
disturb the surrounding lipid organization and promote a tilted
orientation. Finally, another motif for cholesterol binding is the
tetrapeptide YIYF (Epand, 2006; Kuwabara and Labouesse, 2002),
which is frequently found near the end of transmembrane helices
of sterol-sensing domains. Sterol-sensing domains comprise five
transmembrane helices and are found in several proteins involved in
cholesterol homeostasis, such as NPC1, HMG-CoA reductase and
SCAP (Epand et al., 2010). The scheme below shows cholesterol
embedded within the membrane phospholipid bilayer and
neighboring a typical transmembrane helical domain of an integral
membrane protein (type II topology). The positions of potential
cholesterol binding motifs (CARC, R-W-L, CRAC, YIYF) within
SNAREs as listed in Fig. 2B are indicated. This representation
assumes that cholesterol increases the relative thickness of the
bilayer and its order (Sharpe et al., 2010).
N-
CARC
R-W-L
CRAC
YIYF
Cholesterol
-C
sterol transport and distribution (Ikonen, 2008; Maxfield and van
Meer, 2010; Mesmin and Maxfield, 2009). In general, cells
acquire cholesterol through receptor-mediated endocytosis of
LDL or by de novo synthesis in the endoplasmic reticulum (ER)
(Simons and Ikonen, 2000). After LDL internalization, esterified
LDL-cholesterol traffics through the endocytic compartment to
be delivered to late endosomes and lysosomes, where it is
hydrolyzed by lysosomal acid lipase to free the cholesterol
molecule. The majority of late-endosome-derived cholesterol is
then delivered to the plasma membrane, while some of it is
transported to the ER to enable feedback control or undergoes
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esterification for storage in lipid droplets in the form of
cholesteryl esters. The routes and mechanisms by which late
endosome-originating cholesterol reaches the plasma membrane
are not clear, but as shown by our laboratory (Cubells et al., 2007;
Reverter et al., 2014) and others (Kanerva et al., 2013; Urano
et al., 2008), might involve transport through the ER, the transGolgi network (TGN) and recycling endosomes (Fig. 1A). As
outlined below, although recycling endosomes and the TGN
contain much less cholesterol than the plasma membrane,
moderate changes in the levels of cholesterol that are
transported through these compartments appear to have drastic
effects on cellular behavior that is relevant in the context of
migration and invasion.
The physiological importance of LDL uptake and lysosomal
processing is underscored by genetic defects that cause familial
hypercholesterolemia, as well as Niemann Pick type C (NPC)
disease, a lethal condition that is characterized by intracellular
accumulation of unesterified cholesterol in late endosomes and
lysosomes. Niemann Pick C1 protein (NPC1) and Niemann Pick
type C2 protein homolog (NPC2) bind to late-endosome-associated
cholesterol and facilitate its export from late endosomes (Ikonen,
2006; Vance and Karten, 2014). Mutations in the genes encoding
NPC1 or NPC2 inhibit the egress of cholesterol from late
endosomes (Klein et al., 2006) and reduce its delivery to the
Golgi, plasma membrane and recycling endosomes (Cubells et al.,
2007; Kanerva et al., 2013; Reverter et al., 2014; Urano et al.,
2008). Together, this triggers a dysfunction of membrane
trafficking, which is associated with cardiovascular, neurological
and lysosomal storage diseases (Cortes et al., 2013; De Matteis and
Luini, 2011; Ikonen, 2006; Maxfield and Tabas, 2005). (Pre)lysosomal cholesterol export most likely involves the transfer of
cholesterol from luminal NPC2 to membrane-associated NPC1,
followed by insertion of the aliphatic side chain of cholesterol into
the (pre-)lysosomal membrane (Infante et al., 2008; Wang et al.,
2010). Although subsequent cholesterol transfer to other sites is not
fully understood, non-vesicular transport mediated by cytosolic
cholesterol-binding proteins and vesicular pathways exist
(Fig. 1B,C). In addition, membrane contact sites between late
endosome or lysosomes and other compartments, such as the ER,
appear to enable cholesterol transfer (Alpy et al., 2013; Chang
et al., 2006; Ikonen, 2008; Rowland et al., 2014; van der Kant and
Neefjes, 2014).
It should be noted that in some cell types, in particular
hepatocytes and steroidogenic cells, uptake of cholesterol from
high-density lipoprotein (HDL) can significantly contribute to
cholesterol homeostasis. This is mediated by the scavenger
receptor class B member 1 (SRB1, also known as SR-BI), an
integral membrane protein that is highly expressed in liver and
steroidogenic tissues. SRB1 facilitates the incorporation of HDLderived cholesteryl esters through a process called selective
uptake (Leiva et al., 2011). This is not associated with HDL
particle internalization through endosomal or lysosomal pathways,
but involves the binding of HDL to SRB1 at the cell surface,
followed by passive diffusion of HDL-derived cholesteryl esters
into the plasma membrane. Internalized cholesteryl esters are
then rapidly hydrolyzed. Consequently, HDL-derived cholesterol
quickly enters other sites, such as the ER and recycling endosomes,
with kinetics that are much faster than for trafficking of LDLderived cholesterol (Heeren et al., 2006; Leiva et al., 2011).
Therefore, cholesterol generated by this means can rapidly
affect the distribution of cholesterol-sensing SNAREs in these
compartments (see below).
Journal of Cell Science
COMMENTARY
COMMENTARY
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
A
B
ccp
Caveolae
1
Uptake
LDL
HDL
chol-rich
EE/SE/RE
Efflux
4
LE
Lys
9
Degradation
6
5
2
Sorting
13
3
CE
LE/MVB
ER
LD
C
10
8
TGN
Storage
LDL
7
7
Golgi
Esterification
CE
ER
Biosynthesis
LE
12
11
Nucleus
Key
Clathrin
MLN64
ORP5
NPC2
Rab8
AnxA6
Cholesterol
Hrs
NPC1
Rab7
Rab9
STX6
SNARE proteins on endomembranes interact with
cholesterol
In a recent comprehensive survey using photoreactive sterol
probes in combination with quantitative mass spectrometry,
Hulce and co-workers identified over 250 cholesterol-binding
proteins, including several SNARE proteins (Hulce et al., 2013).
More than 60 SNAREs have been identified from yeast and
mammals, and they are known to be crucial components of
protein complexes that drive membrane fusion in secretory and
endocytic pathways (Jahn and Scheller, 2006). Site-specific
interaction and pairing of SNAREs on target membranes (tSNAREs) with SNAREs on vesicles (v-SNAREs) facilitates
tethering, docking and fusion of distinct vesicle-mediated
transport events. This complex and multifactorial process
determines the efficiency and speed of delivery of secretory
vesicles. Although SNAREs were originally classified as v- and tSNAREs according to their localization, they can be structurally
distinguished as Q or R types (Hong, 2005; Hong and Lev, 2014;
Südhof and Rothman, 2009) and further segregated into four
subfamilies. These are the syntaxin subfamily (Qa), the S25N
(Qb) and S25C (Qc) subfamilies and members of the VAMP
subfamily, which are all R-type SNAREs. All syntaxins (except
STX11) and VAMPs are integral membrane proteins and contain
a transmembrane domain. SNAP23, SNAP25 and SNAP29 are all
members of both the Qb and Qc subfamilies and contain two
tandem SNARE motifs, but lack a transmembrane.
Strikingly, from the 38 SNAREs found in humans (Hong and
Lev, 2014), 11 have been shown to interact with cholesterol
(Fig. 2A). However, little structural information on cholesterol–
protein complexes is available, and despite the fact that crystal
structures of single or complexed SNAREs exist, information
regarding their interaction with cholesterol is scarce. One
exception is the relatively well-characterized interaction between
VAMP2 and cholesterol. Here, site-directed spin labeling,
continuous wave and pulsed electron paramagnetic resonance
spectroscopy have shown that a significant conformational change
takes place in the transmembrane domain of VAMP2 upon
cholesterol binding, which is thought to support lipid mixing and
eventually membrane fusion (Tong et al., 2009).
However, only one of the cholesterol-binding SNAREs is
found at the plasma membrane, the t-SNARE STX4 and its
partner SNAP23, which binds to cholesterol through
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Journal of Cell Science
Fig. 1. Overview of cholesterol trafficking pathways. (A) Cellular cholesterol transport pathways. (1) Cholesterol is delivered to early (EE), sorting (SE) and
recycling endosomes (RE) by either endocytosis of LDL through clathrin coated pits (ccp), or selective cholesteryl ester (CE) uptake by SRB1 from HDL in
cholesterol-rich (chol-rich, e.g. caveolae) plasma membrane domains (shown in dark gray). (2) LDL is then transported to late endosomes/multivesicular bodies
(LE/MVBs), where CEs are hydrolyzed. Free cholesterol is then distributed to other sites (see below), whereas the LDL particle is degraded in lysosomes (Lys)
(3). In addition, recycling of cholesterol from EEs, SEs and REs (4) or the LE/MVB (5) to the plasma membrane, or its retrograde transport from EEs, SEs and
REs to the TGN can occur (6). LDL-derived cholesterol in LE/MVBs is distributed to the ER for re-esterification and storage in lipid droplets (LD) (7), to the Golgi
and TGN (8), is exported (efflux) to the plasma membrane (9) and sent to mitochondria (10). Cholesterol synthesized in the ER can be transported to the plasma
membrane directly (11) or through the Golgi (12, 13). (B,C) Vesicular and non-vesicular transport routes of LDL- and HDL-derived cholesterol through late
endosomes. NPC1 in the limiting membrane of late endosomes and lysosomes cooperates with multiple vesicular and non-vesicular cholesterol export pathways.
NPC2 delivers cholesterol to NPC1, which inserts lumenal LDL-cholesterol to the limiting late endosomal and lysosomal membrane. (B) LDL-cholesterol, but only
very little HDL-derived cholesterol (indicated by the dotted line), is delivered to late endosomes. Cytoplasmic carrier proteins facilitate LDL-cholesterol export
from late endosomes and lysosomes. This includes oxysterol-binding protein-related protein 5 (ORP5), which possibly transfers cholesterol through membrane
contact sites to the ER. Hrs/VPS27, a member of the ESCRT, might also be involved in NPC1-dependent late endosome-cholesterol export to the ER (dotted line)
(Du et al., 2012). Transport to mitochondria involves MLN64 (Charman et al., 2010). (C) Alternatively, NPC1 might deliver cholesterol by vesicular membrane
transport that requires Rab proteins (Rab7, Rab8, Rab9), SNAREs (STX6) and possibly annexin A6 (AnxA6) (see text for further details).
COMMENTARY
A
0
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
50
Amino acid
STX4
100
150
Ha
Hb
200
250
Hc
300
350
TM
SNARE
STX5
STX7
STX12
STX18
STX6
STX8
STX10
VTI1B
VAMP2
VAMP3
Key
CRAC motif
CARC motif
R-W-L motif
Y/W in linker
B
SNARE domain
Linker
Transmembrane domain
N-
C
Polybasic
STX4
297
STX5
355
STX7
261
STX12
276
STX18
335
STX6
255
STX8
236
STX10
249
VTI1B
232
VAMP2
116
VAMP3
100
CARC motif
R-W-L
CRAC motif
Fig. 2. Cholesterol-binding motifs in
transmembrane and juxtamembrane domains of
cholesterol-binding SNARE proteins. (A) Schematic
representation of cholesterol-binding human SNARE
proteins showing the position and type of cholesterolbinding motifs in the juxtamembrane or transmembrane
domains (see Box 1). Two SNARE proteins that bind to
cholesterol have not been included in this figure; the first
is Sec22a, an unusual t-(R)-SNARE protein with the
yeast homolog being located at the ER–Golgi interface
and containing a longin domain and a single
transmembrane domain similar to the human Sec22b
isoform. In contrast, the human Sec22a isoform
contains four transmembrane domains with several
CRAC and CARC motifs, but lacks SNARE domain
homology, questioning its classification as a SNARE.
The second is SNAP23, which binds to cholesterol by
means of palmitoylation of cysteine residues in the
linker region between the two SNARE domains. TM,
transmembrane domain. (B) Superposition of the
juxtamembrane regions showing parts of the
transmembrane, linker and SNARE domain of human
cholesterol-binding SNAREs. The sequences (SNARE
database: http://bioinformatics.mpibpc.mpg.de/snare/)
corresponding to the C-terminal region for each protein
reveal the presence of a basic cluster within the linker
region that connects the SNARE and the
transmembrane domain, especially in STX4, STX7 and
STX12. The location of the potential cholesterol-binding
motifs CRAC, CARC and R-W-L within SNAREs (see
Fig. 2B) are indicated with red, orange and blue boxes,
respectively. (C) Model depicting CARC, R-W-L and
other cholesterol-binding motifs (see Box 1) that
contribute to the formation of a cholesterol-enriched
ring-raft in the outer leaflet of the fusion pore, which in
turn would stabilize the local membrane curvature for
membrane fusion.
C
Cholesterol ring-raft
Vesicle
palmitoylated cysteine residues located in the linker region
between the two SNARE domains (Salaün et al., 2005) (Table 1).
Furthermore, several of the SNAREs that are capable of binding
to cholesterol are not known to function in a cholesterol-sensitive
manner, but represent SNAREs that are predominantly found on
endomembranes, often shuttling between locations, such as late
endosomes and recycling endosomes, as well as the Golgi. Thus,
the potential interaction between cholesterol and these SNAREs
on endomembranes could point to an exciting new molecular
pathway that couples local cholesterol levels in endosomal
membranes and the Golgi with dynamic changes in SNARE
location and thereby their function in cell dynamics and behavior.
In line with 70–80% of cellular cholesterol being found at the
plasma membrane, several studies have identified a crucial role
for cholesterol in inducing and stabilizing the clustering of
SNAP23 and STX4, which is important for membrane fusion
(Chamberlain et al., 2001; Lang, 2007; Predescu et al., 2005; Puri
and Roche, 2006). SNAP23 binds to STX4 in vivo (St-Denis
et al., 1999). Accordingly, together these two SNAREs are
involved in the fusion of transferrin-containing recycling vesicles
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with the basolateral membrane (Leung et al., 1998), as well as
surface delivery of newly synthesized proteins from the Golgi to
the apical membrane in Madin–Darby canine kidney (MDCK)
cells (Lafont et al., 1999). Although we and others confirmed that
these SNAREs, similar to STX1 or SNAP25, concentrate within
clusters at the plasma membrane (Reverter et al., 2011; Sieber
et al., 2006; Sieber et al., 2007), the underlying causes that
promote STX4–SNAP23 clustering are not fully understood.
Given the high cholesterol content at the plasma membrane and
its membrane-condensing effect, which increases membrane
order and rigidity (Lingwood and Simons, 2010), the
physicochemical properties of cholesterol probably play a major
role in the formation of SNARE microdomains at the plasma
membrane (van den Bogaart, 2013). In addition, factors that
promote SNARE clustering at the plasma membrane include
their partitioning into cholesterol-enriched membrane rafts,
competition with cholesterol for solvation by bulk lipids, and
electrostatic protein–lipid interactions. Furthermore, the clustering
of plasma-membrane-associated proteins, including the SNAREs
listed above, might be promoted by protein–protein interactions
Journal of Cell Science
SNAREs
COMMENTARY
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
Table 1. Distinctive properties of cholesterol-binding SNARE proteins
SNARE
Type
Location
Cholesterol
selectivea
Cholesterol
sensitivea
STX4
STX5
STX7
STX12
STX18
STX6
STX8
STX10
VIT1B
VAMP2
t-(Qa)
t-(Qa)
t-(Qa)
t-(Qa)
t-(Qa)
t-(Qc)
t-(Qc)
t-(Qc)
v-(Qb)
v-(R)
PM
Golgi
LE/EE
RE/EE
ER
TGN
LE/EE
TGN/RE
LE/EE
SV
+
2
+
+
+
+
+
+
2
+
2
+
+
2
+
2
2
2
2
2
VAMP3
v-(R)
RE/EE
+
2
Cholesterol-binding motifs
CRAC
CARC
R-W-L
Otherb
TMc
Reference
+
23
17
23
23
17
19
19
17
20
22
Chen and Scheller, 2001
Laufman et al., 2011
Mullock et al., 2000
Prekeris et al., 1998
Iinuma et al., 2009
Bock et al., 1997
Antonin et al., 2000
Ganley et al., 2008
Pryor et al., 2004
Chamberlain and Gould,
2002
McMahon et al., 1993
+
*
*
+
+
+
+
+
+
+
+
+
+
*
20
(Zilly et al., 2011), homotypic interactions between the
SNARE motifs (Sieber et al., 2006), heterotypic protein–
protein interactions (Yang et al., 2006) and anchoring to the
cortical cytoskeleton (Low et al., 2006; Torregrosa-Hetland et al.,
2011).
Nevertheless, the extent to which cholesterol-mediated plasma
membrane clustering of SNAREs contributes to their overall
distribution remains unclear. The reported effects of cholesterol
on SNARE clustering are predominantly based on extraction of
cholesterol from cells under non-physiological conditions with
methyl-b-cyclodextrin, which is known to cause a partial to
complete disintegration of plasma membrane clusters that are
enriched in STX1, SNAP25, STX4 and/or SNAP23 (Low et al.,
2006; Predescu et al., 2005; Reverter et al., 2011). Moreover,
within a single cell type, cholesterol extraction can completely
disrupt plasma membrane clusters of a particular SNARE,
whereas other plasma-membrane-associated SNARE clusters
remain unaffected, suggesting that the extent of cholesterolmediated clustering differs among SNAREs and cell types. The
underlying mechanisms have yet to be resolved, but certain
cholesterol pools located in specific microdomains might be more
accessible and susceptible to depletion by experimental protocols,
which could lead to a sequential disruption of membrane
microdomains with variations in their SNARE and cholesterol
content.
The late endosomal compartment is linked to cell migration
The late endosomal compartment is not only central to cholesterol
homeostasis (see above), but recent data are beginning to unravel
how late endosomes and lysosomes influence the dynamics of
adhesions between cells and the ECM and movement of cells in
both two- and three-dimensional microenvironments (Rainero
and Norman, 2013). For instance, the endosomal sorting
complexes required for transport (ESCRT), which regulate
delivery of cargo and membrane transport along the endocytic
pathway, control the transit of the tyrosine kinase Src from late
endosomes to focal adhesions, adhesion structures required for
cell–ECM interactions, at the plasma membrane. At the cell
surface, Src is involved in mediating the disassembly and
turnover of cell–ECM interactions, which are required for cell
migration (Tu et al., 2010). Particularly relevant for cancer
metastasis, oncogenic v-Src kinase is located in perinuclear
structures that resemble late endosomes, and it translocates to the
plasma membrane to mediate cell transformation (Fincham et al.,
1996). Along these lines, in pancreatic and ovarian cancers,
Rab25 together with the chloride intracellular channel 3 (CLIC3)
drives invasiveness by recycling of a5b1 integrin from late
endosomes to the plasma membrane (Dozynkiewicz et al., 2012).
The protein machinery that controls the fusion of late
endosome and lysosomal compartments with the plasma
membrane is now relatively well documented and involves
syntaxin 7 (STX7), STX8, vesicle-associated membrane protein 7
(VAMP7) and VAMP8, which are all members of the SNARE
protein family (Kent et al., 2012; Luzio et al., 2010; Luzio et al.,
2014; Pryor et al., 2004). Chavrier and co-workers have
established that VAMP7 and the exocyst complex deliver a late
endosomal pool of membrane type 1 metalloproteinase (MT1MMP, also known as MMP14) to the cell surface for ECM
proteolysis, which is crucial for invasive migration (Steffen et al.,
2008). Earlier steps in this pathway might involve CLIC3
(Macpherson et al., 2014). Later trafficking events probably
require the actin cytoskeleton, as following its transit from Rab7positive endosomes to the plasma membrane, MT1-MMP is
retained in invasive protrusions by direct tethering to F-actin (Yu
et al., 2012).
In addition, besides these SNARE-dependent trafficking events
that emanate from late endosome and drive cell migration,
distribution of LDL-derived cholesterol from lysosomes and late
endosome to other cellular sites has now been recognized to also
trigger trafficking events that promote cell migration. Recently,
Ikonen and co-workers identified the delivery of LDL-derived
cholesterol from late endosomes to focal adhesions (Kanerva
et al., 2013). As outlined above, cholesterol is essential for the
functioning of SNAREs at the plasma membrane and one could
envisage that the compromised motility and function of late
endosome and lysosomes in lysosomal storage diseases such as
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Journal of Cell Science
SNARE type [t-(Qa), t-(Qc), v-(Qb), v-(R)] and subcellular location are given. EE, LE, RE, early, late or recycling endosomes, PM (plasma membrane), SV
secretory vesicles. The presence of cholesterol-binding motifs (CRAC, CARC or R-W-L) in transmembrane (TM) domains are given. aCholesterol selective
indicates whether proteins selectively bind a trans-sterol probe but not a non-steroidal neutral lipid probe; cholesterol sensitive indicates whether proteins show
competition with unlabeled cholesterol (Hulce et al., 2013). b+, presence of undefined cholesterol-binding motif (i.e. tilted peptide); *, presence of aromatic (Y/W)
residues in the linker region between the SNARE and transmembrane domains. cThe number of hydrophobic residues in the transmembrane domain according
to: http://www.ch.embnet.org/software/TMPRED_form.html.
NPC – which is caused by accumulation of cholesterol and/or
sphingolipids (reviewed in detail in Fraldi et al., 2010; Ikonen and
Hölttä-Vuori, 2004; Platt et al., 2012) – interferes with the
functioning of those SNAREs that are localized in the late
endosome compartment and are relevant for cell migration
(Ganley et al., 2008).
Loss of NPC1 interferes with cholesterol-sensitive vesicle
formation in the Golgi
By using NPC1-mutant-like cell models that accumulate
cholesterol in the late endosomes, by pharmacological means
(treatment with U18666A) or annexin A6 overexpression, which
inhibits NPC1 activity possibly through direct protein–protein
interaction (Cubells et al., 2007), we initially showed that
blocking egress of cholesterol from late endosomes interfered
with cholesterol-dependent caveolin-1 transport from the Golgi to
the cell surface, thereby reducing the number of caveolae
(Cubells et al., 2007). We speculated this to reflect earlier data
from in vitro studies showing that precisely balanced cholesterol
levels are essential to drive vesicle formation from the Golgi
(Stüven et al., 2003). Although cholesterol depletion inhibits the
formation of secretory vesicles from the TGN (Wang et al.,
2000), elevated cellular cholesterol stimulates vesicle formation
and the dispersal of Golgi-derived vesicles (Grimmer et al.,
2005). Efforts to identify the cholesterol-sensitive factor that is
involved in this process found that cytoplasmic phospholipase A2
(cPLA2) translocates to the Golgi upon elevation of cellular
cholesterol (Grimmer et al., 2005). Based on these data and the
fact that cPLA2 converts phospholipids into lysophospholipids
that have an inverted-cone shape, the authors hypothesized that
cPLA2 contributes to Golgi membrane deformations that are
required for the formation of secretory vesicles, as
pharmacological inhibition of cPLA2 has been shown to inhibit
cholesterol-induced vesiculation at the Golgi (Grimmer et al.,
2005).
In further support of this hypothesis, our follow-up studies
revealed that a decrease in the availability of cholesterol in the
Golgi of NPC1-mutant-like cells upon using pharmacological
treatment with U18666A or annexin A6 overexpression perturbed
the translocation to and activity of cPLA2 at the Golgi (Cubells
et al., 2008). We speculated that inhibition of cholesterol egress
from late endosomes leads to a depletion of Golgi cholesterol,
therefore disrupting cholesterol-dependent cPLA2 recruitment
and vesiculation events at the Golgi (Cubells et al., 2008). This
would interfere with cholesterol delivery to the cell surface along
exocytic pathways that originate at the Golgi. As the plasma
membrane undergoes rapid and continuous turnover, the lack of
cholesterol replenishing the cell surface pool would be followed
by the disintegration of STX4 and SNAP23 clusters that form at
cholesterol-enriched plasma membrane microdomains. Consistent
with this model, pharmacological inhibitors of cPLA2 strongly
reduced the localization of SNAP23 and STX4 at the plasma
membrane (Reverter et al., 2011). Moreover, depletion of the
cPLA2 product, arachidonic acid, has been shown to inhibit STX4
from forming SNARE complexes (Connell et al., 2007).
Arachidonic acid induces the so-called ‘syntaxin-opened’
conformation that is required for SNARE assembly (Connell
et al., 2007; Rickman and Davletov, 2005), further supporting the
involvement of cPLA2 in the modulation of syntaxins that
participate in SNARE trafficking and assembly.
Most strikingly, we found that although clusters of SNAP23 and
STX4 at the plasma membrane disintegrated in NPC1 mutant-like
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Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
cells, both SNAP23 and STX4 accumulated together with caveolin1 in Golgi membranes, which as we shown, is associated with an
increased formation of SNAP23–STX4-containing t-SNARE
complexes in the Golgi (Reverter et al., 2011). Consequently, the
accumulation and mislocalization of SNAP23–STX4 complexes in
the Golgi of these NPC1-mutant-like cells prevented SNAP23–
STX4 from promoting the secretion of fibronectin, an ECM
component that binds to cell adhesion receptors, at the plasma
membrane. Mislocalization and loss of SNAP23–STX4 function
upon cholesterol depletion might also occur in other STX4–
SNAP23-dependent events that are relevant for cancer cell
migration, for instance the association of STX4–SNAP23 with
VAMP7, which is required to deliver MT1-MMP to invadopodia
for ECM degradation in invasive cancers (Williams et al., 2014).
Thus, the depletion of cholesterol from the Golgi and plasma
membrane owing to its accumulation in late endosomes not only
interferes with the localization and organization of some
SNAREs at the plasma membrane, but also impacts on the
dynamics of SNARE assembly and disassembly on intracellular
membranes, such as at the Golgi and transport pathways
originating from the Golgi (Reverter et al., 2011; Reverter
et al., 2014).
Golgi-localized cholesterol regulates SNARE-dependent
integrin trafficking
Based on the studies discussed above, it is tempting to speculate
that the mislocalization of SNAP23–STX4 upon NPC1 inhibition
is only an example of the broader impact a cellular imbalance of
cholesterol has on endomembrane SNAREs, including those
involved in post-Golgi exocytic pathways through the TGN and
recycling endosome compartments and those relevant for cell
migration.
Integrins are cell adhesion receptors; they are composed of a
and b subunits that bind to the ECM and enable cells to migrate.
To ensure forward movement, integrins are constantly
internalized and recycled for their redistribution at the leading
edge (Caswell and Norman, 2008; Jones et al., 2006; Pellinen and
Ivaska, 2006). Both cholesterol and several SNAREs have been
implicated in the molecular machinery that is responsible for the
endocytic and exocytic trafficking of integrins. As such, the
cholesterol content of the plasma membrane controls signaling
events that are mediated by aVb3 integrins (Green et al., 1999),
including focal adhesion kinase (FAK) and mitogen-activated
protein kinase (MAPK) signaling pathways, and cell adhesion
and migration on fibronectin-coated substrates (Ramprasad et al.,
2007). In this context, the ability of SNAREs to bind cholesterol
in specific cellular sites could contribute to the establishment of
functional links between cholesterol and integrin localization and
function. These cholesterol-binding SNAREs include VAMP2,
VAMP3 (also known as cellubrevin), STX3, STX4 and SNAP23,
which all participate in the recycling of b1 integrin, and VAMP3
and STX6, which determine the levels of cell-surface-associated
a5b1 integrin and of FAK (Hulce et al., 2013; Riggs et al., 2012;
Tiwari et al., 2011) (see also Fig. 2A and Table 1).
Cholesterol-sensitive shuttling of STX6 between the TGN and
recycling endosomes determines integrin recycling and
migratory behaviour of cells
Among the aforementioned v-SNAREs, VAMP3 contains a
cholesterol-interacting R-W-L motif (Fig. 2B). VAMP3 is highly
similar to VAMP2 and has been shown to bind to cholesterol in
exocytic pathways (Table 1). Besides its similarity to VAMP2,
Journal of Cell Science
COMMENTARY
VAMP3 also displays a significant sequence similarity to
VAMP4, which might contribute to the ability of both VAMPs
to bind the t-SNAREs STX6, STX16 and VIT1A, which function
at the trafficking interface between the TGN, recycling endosome
and the plasma membrane. However, VAMP4 does not appear to
bind cholesterol, but contains an E/DxxxLL motif that allows
interaction with adaptor protein-1 in the TGN (Peden et al.,
2001). These differential binding properties might enable
VAMP3 and VAMP4 to participate in different trafficking
pathways that intersect at the TGN, where they interact with
their common t-SNARE partner STX6.
As discussed above, both VAMP3 and VAMP4 interact with
the t-SNARE STX6, which, at steady state, is predominantly
localized at the TGN where it binds to resident molecules through
its SNARE motif (Bock et al., 1997; Wendler and Tooze, 2001)
and Habc domain (Abascal-Palacios et al., 2013; Pérez-Victoria
et al., 2010). Besides VAMP4, this includes the Golgi SNARE
protein GS32 (also known as SNAP29) and the mammalian Golgi
Sec1p-like protein VPS45, both of which are known to be
involved in TGN trafficking (Watson and Pessin, 2000). STX6
participates in various membrane fusion events with different
SNARE complexes. Upon completion of membrane fusion, a
tyrosine-based sorting motif (YGRL, position 140–143) located
between the N-terminal domain and the SNARE motif is
activated, which results in its re-sorting to the TGN (Jahn and
Scheller, 2006; Jung et al., 2012). Importantly, STX6 binds to
cholesterol (Hulce et al., 2013) and has been previously
implicated in cholesterol transport; it contributes to the delivery
of late-endosome-derived cholesterol to the ER via the TGN
(Urano et al., 2008) and of lipids and proteins that are required for
caveolae endocytosis to the plasma membrane (Choudhury et al.,
2006). This work has shown that inhibition of STX6 leads to a
reduction of caveolin-1 and caveolae at the cell surface, lending
further support for a model by which STX6 regulates secretory
pathways in a cholesterol-sensitive manner. This ability of STX6
to regulate the transport of cholesterol-rich domains to and from
the cell surface could determine the cell surface levels of a5b1
integrin and FAK, and so modulate focal adhesion sites and
directional migration on fibronectin-coated substrates (Tiwari
et al., 2011).
Taken together, cholesterol is linked to the localization and
function of STX6 and other SNAREs that are predominantly
located at the TGN, the TGN–recycling-endosome boundaries
and recycling endosomes. Using an array of NPC1 mutant
models, we were able to provide novel insights into how
cholesterol pools at the Golgi–endosomal boundaries regulate
cell migration, namely through altering the assembly and
disassembly of complexes between STX6 and VAMP3 or
VAMP4, respectively (Reverter et al., 2014). Mechanistically,
we found that a decrease in the cholesterol level at the Golgi
perturbed the trafficking between recycling endosomes and TGN
and triggered the accumulation of STX6 in VAMP3- and Rab11containing recycling endosomes. This correlated with a reduced
cell surface expression of integrins in NPC1 mutant models and
resulted in impaired cell migration and invasion in two- and
three-dimensional environments. Support for endomembrane
cholesterol levels altering SNARE localization and function has
been provided by the fact that prolonged treatment of NPC1
mutant cells with LDL, which causes its overspill into late
endosomes, thereby enabling LDL-cholesterol to bypass the
NPC1 mutation and to enter the TGN at later time points, induced
the targeting of STX6 back to the TGN. By contrast, incubation
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
of wild-type cells with HDL-cholesterol, which rapidly enters
recycling endosomes, but not the TGN, within 30 min after
binding of HDL to cell surface receptors, results in a
relocalization of STX6 from the TGN to recycling endosomes
(Reverter et al., 2014). Thus, the rapid and local elevation of
cholesterol levels in recycling endosomes of wild-type cells
observed after HDL incubation appears to increase the ability of
STX6 to interact with VAMP3 in recycling endosomes.
Further support for a function of STX6 as a cholesterol-sensitive
endomembrane SNARE comes from its structure; this syntaxin
contains an R-W-L motif (position 233–241) (Fig. 2B), as well as a
polybasic cluster in the juxtamembrane region that has homology
to STX4, STX7 and STX12. This polybasic region is conserved
and also found in STX1A, STX1B, STX2, STX3, STX17, STX20
and STX16 (Murray and Tamm, 2011). In contrast, this positively
charged region appears to be less prevalent in syntaxins that are
required for early steps in membrane trafficking, such as STX18 in
the ER or STX5 in the Golgi. Interestingly, the syntaxins with the
most positive charges localize to the plasma membrane in
microdomains that are characterized by a high degree of negative
charge density and are enriched in cholesterol (Murray and Tamm,
2011). Thus, the spatiotemporal localization and function of the
syntaxins that have cholesterol-binding motifs and polybasic
clusters, such as STX6, STX4, STX7 and STX12, might be
highly responsive to fluctuations in cholesterol levels at either the
plasma membrane or intracellular compartments.
The drastic changes in STX6 localization we observed in
NPC1-mutant cells and upon incubation with lipoproteins suggest
that STX6 trafficking between TGN and recycling endosome and
its compartment-specific interaction with the v-SNARES
VAMP3 and VAMP4 is controlled by its ability to sense
cholesterol levels in the TGN and/or recycling endosomes,
thereby possibly regulating cell migration through STX6dependent trafficking of the integrins aVb3 and a5b1. NPC1mediated reduction of cholesterol in TGN membranes or selective
elevation of recycling endosome-localized cholesterol could
promote the translocation of STX6 to these membranes and so
increase its ability to interact with VAMP3 (Reverter et al.,
2014). In fact, recycling endosomes are the main intracellular
cholesterol repository compartments in a number of cell types,
including Chinese hamster ovary and non-polarized hepatoma
HepG2 cells, fibroblasts and human B lymphocytes (Hao et al.,
2002; Hsu and Prekeris, 2010; Maxfield and McGraw, 2004).
Although other alternative pathways might exist, our findings
point to a link between cholesterol-sensitive SNAREs and the
final steps in integrin recycling relevant for wound healing and
cancer cell migration (Fig. 3). Earlier work has implicated
cholesterol in the formation of signaling complexes that contain
aVb3, CD47 and G-proteins (Green et al., 1999) and in the
control of cell adhesion and migration onto fibronectin. This
possibly requires Rab11, which modulates cholesterol transport
and homeostasis (Hölttä-Vuori et al., 2002), and facilitates the
recycling of b1 integrin (Powelka et al., 2004). Further support
for this notion comes from an increased cholesterol requirement
for invasion in MDA-MB-231 breast cancer and A431
epidermoid carcinoma cells (Freed-Pastor et al., 2012); both
cell models are highly relevant in this context as enhanced
integrin recycling therein drives aggressive cancer cell behavior
(Muller et al., 2009). It has yet to be demonstrated whether these
observations are based on some SNAREs functioning as
cholesterol sensors in cancer. Indeed, STX6 overexpression
increases cell migration, and its expression is elevated in breast,
1077
Journal of Cell Science
COMMENTARY
COMMENTARY
Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459
Clathrincoated pit
Fibronectin
secretion
Caveolae
STX6
STX4
SNAP23
VAMP8
Rab5
Rab11
2
STX12
VAMP3
2
Rab4
RE
EE
1
Rab7
Golgi
complex
LE
STX6
STX16
VTI1A
VAMP3
STX7
VTI1B
STX8
VAMP7
VAMP8
Key
Clathrin
Rab9
Cholesterol
NPC1
NPC2
STX6
STX16
VTI1A
VAMP4
Integrin α5β
Integrin αVβ3
liver and prostate cancers (Riggs et al., 2012), which all have
established links to cholesterol homeostasis.
Furthermore, in breast cancer cells, SNAP23 and STX12, which
also bind to cholesterol in the plasma membrane and recycling
endosomes, are required for the delivery of Src, epidermal growth
factor receptor (EGFR) and b1 integrin to cholesterol-rich
invadopodia for cell invasion. Interestingly, b1 integrin affects
the interaction between SNAP23 and STX12 and the formation of
the SNARE-dependent Src–EGFR–b1-integrin complex (Williams
and Coppolino, 2014). In this context, cholesterol-dependent
trafficking mechanisms appear to be essential (Caldieri and
Buccione, 2010), and cholesterol-binding proteins such as
caveolin-1 (Murata et al., 1995) actively participate in the
transport of cholesterol, as well as facilitating its interaction with
integrins and their activation (Parton and del Pozo, 2013).
Conclusions and perspectives
As discussed above, cholesterol levels at late endosomes and the
Golgi modulate the localization and activity of a subset of
cholesterol-sensing SNARE proteins that regulate fibronectin
secretion, integrin recycling and cell migration.
The underlying molecular mechanisms await further
investigation. Cholesterol levels in the TGN and recycling
endosome are much lower than those in the plasma membrane.
Together with the drastic experimental procedures (e.g. treatment
with methyl-b-cyclodextrin) required to reduce membrane order
at the plasma membrane (Reverter et al., 2014), this might
indicate that moderate changes in cholesterol levels are
insufficient to significantly alter membrane fluidity within the
TGN or recycling endosome subcompartments to affect the
affinity of SNAREs towards membranes and thus their
1078
distribution. Instead, we envisage that cholesterol transport is
targeted directly to or into the vicinity of SNARE complexes,
possibly by the docking of cholesterol carriers into close
proximity or directly onto endomembrane SNAREs. This could
enable SNARE–cholesterol interaction to impact on the stability
of assembled SNARE complexes within a certain location,
providing the opportunity for these complexes to dissociate and
move along transport pathways to find other interaction partners
elsewhere. However, it is still unknown whether changes in
cholesterol levels affect the three-dimensional structure and
conformation of specific SNARE proteins (as postulated for
VAMP2) (Tong et al., 2009), or whether cholesterol levels and
the lipid environment could interfere with the affinity or
capability of certain SNAREs to cluster and/or remain in a
particular membrane.
Alternatively, rather than directly targeting endomembrane
SNAREs, cholesterol could modulate the activity of Golgitethering factors that are required for SNARE assembly. For
instance, Golgi-associated retrograde protein (GARP) and
conserved oligomeric Golgi (COG) complex regulate the fusion
of endosome-derived, retrograde transport carriers to the TGN.
This process requires the assembly of the t- and v-SNAREs (STX6,
STX16, VIT1A and VAMP4) to facilitate the final steps in the
retrograde transport of recycled receptors or integrins, which enter
the trans-Golgi for re-sialylation, or transport of Golgi-resident
proteins (i.e. TGN38) to TGN–endosome boundaries (Bonifacino
and Hierro, 2011). The ability of GARP, COG and other Golgitethering complexes to promote or prevent the assembly of the vand t-SNAREs mentioned above or possibly the pairing of other
specific v- and t-SNAREs might depend on their spatiotemporal
localization and ability to interact with other cellular SNAREs.
Journal of Cell Science
Fig. 3. Model for how cholesterol-sensitive SNARE complexes in endocytic and exocytic trafficking pathways regulate cell migration. Loss of NPC1
function blocks the exit of cholesterol from the late endosome (LE), which leads to reduced cholesterol levels at the Golgi complex and the plasma membrane. This
triggers the mislocalization of the t-SNARE STX6 from the Golgi complex to recycling endosomes (RE) (1). Increased assembly of the STX6–VAMP3 complex in
recycling endosomes reduces recycling and cell surface expression of aVb3 and a5b1 integrins (2), which negatively impacts on cell migration. Orange arrows
indicate trafficking routes of cholesterol. Blue arrows show integrin trafficking routes. Dotted lines represent cholesterol trafficking routes that are impaired due to loss
of NPC1 function. Black arrows indicate vesicular transport routes from the Golgi to recycling endosomes and the plasma membrane. Cholesterol-rich membrane
domains (dark gray), endocytic routes through clathrin coated pits or caveolae, and several Rab proteins are indicated. SNARE complexes within a particular
compartment are indicated in boxes; the cholesterol-sensitive SNAREs in each compartment are highlighted in orange. EE, early endosomes.
Besides the affinity of tethers to their respective SNAREs, the
avidity of binding, post-translational modifications or as
highlighted here, the amount of cholesterol can influence the
assembly or disassembly of SNAREs. The identified role for
cholesterol in controlling integrin trafficking through SNAREs
could not only be relevant for the ability of cancer cells to move
and spread, but is also important for wound healing in diabetes.
Undoubtedly, further in vivo and in vitro studies will shed more
light on the complex role cholesterol plays in modulating the
molecular mechanisms that enable SNAREs to control membrane
trafficking and fusion events that are relevant for cell migration in
both physiological and pathological contexts.
Acknowledgements
We would like to thank all members of our laboratories, past and present, for their
invaluable contributions and apologize to all those researchers whose work could
not be discussed owing to space limitations.
Competing interests
The authors declare no competing or financial interests.
Funding
This work was supported by the Ministerio de Economı́a y Competitividad
(MINECO) [grant numbers BFU2012-36272 and CSD2009-00016] and Fundació
Marató TV3 [grant number PI042182] (Spain) to C.E.; and The University of
Sydney, Australia [grant numbers U1758, U7007] to T.G., C.R. is thankful to
CONSOLIDER-INGENIO (MINECO) research program for post-doctoral fellowship
[grant number CSD2009-00016]. A.H. is grateful to Carlos III Health Institute [grant
number PI11/00121] and Basque Government [grant number PI2011-26].
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