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Unravelling the orientation of the inositol-biphosphate ring and its dependence on Phosphatidylinositol 4,5-bisphosphate cluster formation in model membranes

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Hypothesis: Inositol phospholipids are well known to form clusters in the cytoplasmic leaflet of the plasma membrane that are responsible for the interaction and recruitment of proteins involved in key biological processes like endocytosis, ion channel activation and secondary messenger production. Although their phosphorylated inositol ring headgroup plays an important role in protein binding, its orientation with respect to the plane of the membrane and its lateral packing density has not been previously described experimentally. Experiments: Here, we study phosphatidylinositol 4,5-bisphosphate (PIP2) planar model membranes in the form of Langmuir monolayers by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry to elucidate the relation between lateral (in-plane) and perpendicular (out-of-plane) molecular organization of PIP2. Findings: Different surface areas were explored through monolayer compression, allowing us to correlate the formation of transient PIP2 clusters with the change in orientation of the inositol-biphosphate headgroup, which was experimentally determined by neutron reflectometry.
A Thickness of tails (green triangles) and headgroups (blue circles) plotted versus surface pressure, together with total thickness of the monolayer (black squares). B Volume fraction of water in the headgroups layer plotted versus surface pressure. C Plot showing the thickness increment of the headgroups layer obtained from the fit versus surface pressure. The linear dependence is shown as a red line. D The calculated values of tilt angle (s) versus surface pressure are shown as black squares. The fit performed using a cos À1 function (Equation (1)) is shown as a red line. The interpolated point (35 mN . m À1 ; 40°) [20-21] is shown in blue. E Sketches representing mixed DPPC:PIP 2 monolayer at the three pressures. The inositol-biphosphate (IP 2 ) ring is depicted in red. The normal to the monolayer (dashed grey line) and the vector connecting C1 and C4 of the inositol-biphosphate (IP 2 ) ring (v 14 , dashed green line) are shown. The tilt angle (s), which is calculated between the normal and v 14 , is shown and depicted as light brown area. F Comparison between out-of-plane structure and in-plane organization of DPPC:PIP 2 mixed monolayers at the three surface pressures. The central panel reports the values of PIP 2 inositol ring tilt angle (s) and tails tilt angle (/) versus area per molecule. Left panel shows the monolayer structure at a low area per molecule value as well as the graphical definition of s and /. Right panel reports a sketch of PIP 2 cluster in the mixed monolayer at larger areas. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Unravelling the orientation of the inositol-biphosphate ring and its
dependence on phosphatidylinositol 4,5-bisphosphate cluster formation
in model membranes
Andreas Santamaria
a,b,1
, Javier Carrascosa-Tejedor
a,c,1
, Eduardo Guzmán
b,d,
, Nathan R. Zaccai
e,2,
,
Armando Maestro
f,g,
a
Large Scale Structures Group, Institut Laue-Langevin, 71 Avenue des Martyrs, 38042 Grenoble, Cedex 9, France
b
Departamento de Química-Física, Facultad de Ciencias Químicas, Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain
c
Division of Pharmacy and Optometry, University of Manchester, Manchester M13 9PT, United Kingdom
d
Instituto Pluridisciplinar, Universidad Complutense de Madrid, Paseo Juan XXIII 1, 28040 Madrid, Spain
e
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB22 7QQ, United Kingdom
f
Centro de
´sica de Materiales (CSIC, UPV/EHU) - Materials Physics Center MPC, Paseo Manuel de Lardizabal 5, E-20018 San Sebastián, Spain
g
IKERBASQUE—Basque Foundation for Science, Plaza Euskadi 5, Bilbao 48009, Spain
graphical abstract
article info
Article history:
Received 6 June 2022
Revised 3 September 2022
Accepted 18 September 2022
Available online 23 September 2022
Keywords:
Phosphatidylinositol 4,5-bisphosphate
(PIP
2
)
abstract
Hypothesis: Inositol phospholipids are well known to form clusters in the cytoplasmic leaflet of the
plasma membrane that are responsible for the interaction and recruitment of proteins involved in key
biological processes like endocytosis, ion channel activation and secondary messenger production.
Although their phosphorylated inositol ring headgroup plays an important role in protein binding, its ori-
entation with respect to the plane of the membrane and its lateral packing density has not been previ-
ously described experimentally.
Experiments: Here, we study phosphatidylinositol 4,5-bisphosphate (PIP
2
) planar model membranes in
the form of Langmuir monolayers by surface pressure-area isotherms, Brewster angle microscopy and
https://doi.org/10.1016/j.jcis.2022.09.095
0021-9797/Ó2022 The Author(s). Published by Elsevier Inc.
Corresponding authors at: Departamento de Química-Física, Facultad de Ciencias Químicas, Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain (E.
Guzmán); Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB22 7QQ, United Kingdom (Nathan R. Zaccai); Centro de
´sica de Materiales (CSIC,
UPV/EHU) - Materials Physics Center MPC, Paseo Manuel de Lardizabal 5, E-20018 San Sebastián, Spain (A. Maestro).
E-mail addresses: eduardogs@quim.ucm.es (E. Guzmán), nrz20@cam.ac.uk (N.R. Zaccai), armando.maestro@ehu.eus (A. Maestro).
1
These authors contributed equally to this work.
2
Present address: Domainex, Pampisford, Cambridge CB22 3EG, United Kingdom.
Journal of Colloid and Interface Science 629 (2023) 785–795
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science
journal homepage: www.elsevier.com/locate/jcis
PIP
2
headgroup orientation
PIP
2
cluster formation
Neutron reflectometry
Lipid monolayer
Air/water interface
Brewster angle microscopy
neutron reflectometry to elucidate the relation between lateral (in-plane) and perpendicular (out-of-
plane) molecular organization of PIP
2
.
Findings: Different surface areas were explored through monolayer compression, allowing us to correlate
the formation of transient PIP
2
clusters with the change in orientation of the inositol-biphosphate head-
group, which was experimentally determined by neutron reflectometry.
Ó2022 The Author(s). Published by Elsevier Inc.
1. Introduction
Despite inositol phospholipids (IPs) are scarcely present in
eukaryotic cell membranes, their spatial assembly forming docking
sites on the inner leaflet of membranes is crucial for the recruit-
ment of specific proteins from the cytosol [1–3]. The organization
of IPs in clusters is well rationalized due to their electrostatic inter-
action with intracellular divalent cations. Interestingly, recent
experimental [4–5] and numerical [6] studies focused on phos-
phatidylinositol 4,5-bisphosphate (PI(4,5)P
2
or, hereinafter, PIP
2
)
have demonstrated that a purely electrostatic, multivalent ion-
mediated mechanism is enough to induce PIP
2
clustering in biomi-
metic model membranes. The effect of divalent cations on the in-
plane organization of PIP
2
containing membranes was previously
studied by imaging, spectroscopy [5,7] and atomistic molecular
dynamic simulations [6]. The existence of PIP
2
domains was attrib-
uted to the capacity of divalent cations, most predominantly Ca
2+
and Mg
2+
, to interact with PIP
2
headgroups and act as a bridge
between PIP
2
molecules, yielding well-defined clusters. Neverthe-
less, the question of how the orientation of the PIP
2
headgroup
with respect to the membrane correlates with its lateral packing
density in presence of divalent cations was not addressed.
Since PIP
2
is the most abundant phosphoinositide in the plasma
membrane inner leaflet of mammalian cells (1–2 % mol) [3,8–10],
recent research has been focused on elucidating its role in model
membranes [7,11] as well as its interaction with proteins [10,12–
14]. PIP
2
is characterized by a net negative electrostatic charge in
the headgroup, as well as an acyl chain asymmetry with a high
degree of unsaturation. Its phosphorylated inositol ring headgroup
enables specific proteins to bind via electrostatic interaction, trig-
gering or contributing to a plethora of important biological path-
ways, such as the attachment of the cytoskeleton to the plasma
membrane, membrane trafficking, and ion channel and enzyme
activation [3,8–10,15]. Moreover, since PIP
2
is characterized by a
large headgroup to acyl chain ratio, it has an inverted conical
shape, thereby favouring the bending of the membrane into a pos-
itive curvature, i.e. bending the monolayer away from the head-
groups [16]. However, this curvature is often sensed and aided
by the presence of proteins, which can induce pits or tubules for-
mation [13,17–19]. Since the key feature of all these molecular
processes is the interaction with the inositol-biphosphate, unravel-
ling the ring orientation in biological membranes prior to the pres-
ence of proteins is essential. Although molecular dynamic
simulations of PIP
2
suggested a preferred tilt angle of 40°
[3,20–21], defined as the angle between the vector connecting C1
and C4 of the inositol-biphosphate ring and the membrane normal
(See Fig. 1B), there is a lack of experimental validation. Previous
in vitro studies focused primarily on the influence of PIPs on model
membranes and not on determining preferred tilt angles
[11,14,22].
Therefore, given the importance of PIP
2
docking sites, and the
likely influence of preferred headgroup orientations on
membrane-protein interactions, it is essential to elucidate the rela-
tion between lateral (in-plane) and perpendicular (out-of-plane)
molecular organization of PIP
2
. To this end, we designed a simple,
in vitro model based on Langmuir monolayers composed of
30 % mol of PIP
2
and 70 % mol of a zwitterionic lipid (1,2-dipalmi
toyl-sn-glycero-3-phosphocholine, DPPC). Although the overall
PIP
2
concentration in the inner leaflet of the mammalian plasma
membrane is much lower (1% mol), this model reflects the high
local concentration of the PIP
2
present at the docking sites on the
cell membrane [23]. DPPC was chosen because its structure is well
known to form stable monolayers and bears structural similarities
to a large diversity of phospholipids in biological membranes.
Indeed, Langmuir monolayers have been widely used to mimic cel-
lular membranes in vitro [24–30]. The lipid monolayer configura-
tion in a Langmuir trough allows the in situ use of neutron
reflectometry (NR), ellipsometry and Brewster angle microscopy
(BAM). While the latter is used to visualize the in-plane interfacial
organization of lipid monolayers, NR and ellipsometry can be
exploited to obtain information about monolayer composition
and structure in the direction perpendicular to the plane of the
interface. In particular, NR is well suited for the structural study
of planar biomimetic membranes with sub-nanometric resolution.
Besides, as neutrons interact very differently with hydrogen and
deuterium, it was possible to exploit isotopic substitution, using
lipid with hydrogenous and deuterated aliphatic tails, to highlight
structural differences in between the hydrophobic tails and the
polar headgroups (including the associated water molecules) con-
stituting mixed monolayers of DPPC and PIP
2
(DPPC:PIP
2
monolay-
ers). These experiments were crucial for determining the tilt angle
of the inositol-biphosphate ring and its dependence on the lipid
surface pressure (
P
) and area per molecule (A). Since membrane
deformation is known to be modulated through membrane area/-
pressure changes, a broad range of surface pressures was explored
through compression, allowing to find a correlation between the
lateral PIP
2
distribution (which influences transient PIP
2
cluster
formation), and the variation of the PIP
2
tilt angle.
2. Materials and methods
2.1. Chemicals
Hydrogenous 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (h-
DPPC), chain-deuterated DPPC (d62-DPPC) and brain L-
a
-phospha
tidylinositol-4,5-bisphosphate (ammonium salt) PI(4,5)P
2
(PIP
2
)
were purchased as powder from Avanti Polar Lipids (purity > 99%,
Alabaster, AL, USA). Figure S1 shows the lipid molecular structure.
Solutions of 1 mgmL
1
of h-DPPC, d62-DPPC and PIP
2
were pre-
pared in chloroform stabilized with ethanol (purity 99.8%;
Sigma-Aldrich, St. Louis, MO, USA). These solutions were used as
stock solutions in order to prepare mixtures with the desired com-
positions, i.e., h-DPPC:PIP
2
and d62-DPPC:PIP
2
7:3 M ratio, at
0.1 mg
.
mL
1
. Ultra-pure water was generated by passing deionized
water through a Milli-Q unit (total organic content = 4 ppb;
resistivity = 18 m
X
cm, Milli-Q, Merck KGaA, Darmstadt, Ger-
many). D
2
O (99.9% of isotopical purity) was purchased from
Sigma-Aldrich (St. Louis, MO, USA) and used as received. Experi-
ments were performed in HKM buffer (25 mM HEPES pH 7.2,
125 mM potassium acetate, 5 mM magnesium acetate, 1 mM
Dithiothreitol -DTT). HEPES (in solution, 1 M in H
2
O, and powder,
purity 99.5%), potassium acetate (purity 99.0%), magnesium
acetate (purity 99.0%) and DTT (purity 99.0%) were purchased
from Sigma Aldrich (St. Louis, MO, USA).
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
786
2.2. Preparation of Langmuir lipid monolayers: Surface pressure - area
isotherm
The surface pressure (
P
) - area per molecule (A) isotherms were
measured using a Langmuir trough (KIBRON, Helsinki, Finland)
with a maximum area of 166.4 cm
2
. The trough was carefully
cleaned with Decon 90 (Decon Laboratories Ltd, Conway St, UK),
absolute ethanol (BioUltra, for molecular biology, purity 99.8%,
Sigma-Aldrich, St. Louis, MO, USA) and Milli-Q water before filling
it with 120 mL of HKM buffer. Subsequently, the lipid solution with
a concentration of 0.1 mgmL
1
was spread on the clean subphase
using a Hamilton microsyringe with a precision of ± 1
l
L. After the
chloroform was evaporated for about 20 min, the variation of sur-
face pressure during compression was recorded using a contact
probe (paper Wilhelmy plate), at a barrier speed of 8 cm
2
min
1
.
In all the experiments performed, the temperature of the subphase
was maintained at 21.0 ± 0.5 °C.
2.3. Brewster angle microscopy
In situ visualization of the morphology of Langmuir monolayers
at the air/water interface was performed using a Brewster Angle
Microscope Nanofilm EP3 (Accurion, GmbH, Göttingen, Germany)
coupled to the previously described Langmuir trough. In brief, spa-
tial reflectivity modulations are correlated with optically different
monolayer phases depending on lateral ordering and/or lipid pack-
ing density. The instrument was equipped with a 50 mW laser
emitting p-polarized light at a wavelength 532 nm directly focal-
ized on the air/water interface at the Brewster angle (53.1°), a
10x magnification objective and a polarizer which drive the
reflected light to a CCD camera. The spatial resolution of BAM
images was 2
l
m and the field of view 350 275
l
m
2
. Further
analysis of the BAM images was performed by using the software
ImageJ [31]. In short, the relative areas occupied by the condensed
phases were calculated by applying a threshold before processing
the images. The error was determined from the precision of the
threshold and the statistical analysis over several images and sam-
ples. Gaussian distributions were used to fit the experimental areas
obtained. The experiments were performed using HKM buffer as
subphase.
2.4. Neutron reflectometry
NR experiments were performed on FIGARO, a time-of-flight
reflectometer [32–34] at the Institut Laue-Langevin, Grenoble
(France), using two different angles of incidence (h
1
= 0.6°and
Fig. 1. A Cartoon representing a mixed DPPC:PIP
2
Langmuir lipid monolayer spread on a Langmuir trough. Incoming and reflected neutron beams are showed to illustrate the
in-situ characterization by Neutron Reflectometry. BMolecular structure of inositol-triphosphate from PBD 1HFA [12]. The distances between the oxygen atom bound to C1
and the oxygen atoms bound to P4 and P5 are shown as dashed black lines; the relative oxygen atoms are shown as black circles. CSketch representing the one-layer and two-
layer models used to fit the Neutron reflectometry data.
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
787
h
2
= 3.7°). A wavelength resolution of 7% dk/kwas used, yielding a
momentum transfer of 0.01 Å
1
<Q
z
< 0.25 Å
1
, normal to the
interface, and defined as Q
z
¼4
p
=kðÞsin h, where k(from 3 to
13 Å) is the wavelength of the neutron beam. In a typical experi-
ment, Reflectivity (R), defined as the ratio of the intensity of the
neutrons scattered from the air/water interface over the intensity
of the incident neutron beam, is measured in specular conditions
(i.e., the incident angle of the neutron beam is equal to the reflected
angle, denoted as h) as a function of Q
z
. The raw time-of-flight
experimental data at the two angles of incidence were calibrated
with respect to the incident wavelength distribution and the effi-
ciency of the detector yielding the resulting R(Q
z
) profile using
COSMOS [35]. This profile is linked to an in-plane averaged scatter-
ing length density (SLD) distribution perpendicular to the interface,
which is a measure of the coherent scattering cross-section of the
molecular species that constitutes each interfacial layer.
NR experiments were performed using two subphases, provid-
ing different contrasts to neutrons: 100 % D
2
O and a mixture
8:92 v/v%ofD
2
O:H
2
O, known as air contrast matched water
(ACMW), since its scattering length density is equal to the one of
air, which is zero. A Langmuir trough was used to deposit lipid
monolayers as explained above.
3. Results and discussion
3.1. Phase behaviour of DPPC:PIP
2
monolayers: Surface pressure - area
per molecule isotherm and compressional elastic modulus
The benefit of working with lipid monolayers (Fig. 1 A) with
regard to lipid bilayers is that a much large region of the lipids’
interfacial phase diagram can be explored from their surface
pressure-area per molecule (
P
-A) compression isotherm. The evo-
lution of lateral pressure as a function of the average area available
for each lipid molecule provides insightful information about the
lipid phase behavior. Indeed, it allows to distinguish if a lipid
monolayer is in a liquid expanded (LE, fluid-like) phase or in a liq-
uid condensed (LC, gel-like) phase. Furthermore, from the slope of
the
P
-A isotherm it is possible to determine the lateral compress-
ibility of the monolayer, i.e., its mechanical resistance against a
dilational deformation in terms of the compressional elastic mod-
ulus (C
s
1
) of the film (Equation S1).
The
P
-A isotherm at 21 °C of a DPPC:PIP
2
(7:3) mixed mono-
layer and, as reference, the isotherms corresponding to pure DPPC
[36] and pure PIP
2
, all of them deposited on a buffer solution con-
taining divalent cations (Mg
2+
), are shown in Fig. 2 A. The corre-
sponding representation of the compressional elastic modulus
(C
s
1
) versus
P
and A are reported in Fig. 2 Band C, respectively.
Interestingly, the aspect of the DPPC:PIP
2
mixed monolayer iso-
therm shown in Fig. 2 Aappears similar to the one of pure DPPC,
in agreement with previous results by Dietrich et al. [14]: both dis-
play a LE phase at low values of surface pressure (
P
<5mNm
1
)
characterized by a lack of in-plane spatial and orientational organi-
zation of the lipid molecules. Further compression yielded an
increase in lateral density and both monolayers showed a coexis-
tence of the LE phase with a more ordered, condensed state (LC)
that consists of tightly packed lipids. There is, therefore, a LE-LC
transition that can be described as a disordered/ordered phase
transition of a 2D fluid [37]. Here, the LE-LC coexistence region,
whose identification a priori was not straightforward from the
visualization of the
P
-A isotherm of both monolayers, was evident
from the appearance of a minimum in the C
s
1
profile. This mini-
mum has been already reported for DPPC monolayers spread onto
both pure water [33] and buffer (rich in Mg
2+
)[36] subphases. Fur-
ther compression yielded a LC phase for both DPPC and DPPC:PIP
2
monolayers. Finally, the emergence of a pseudo-plateau in the iso-
therm of the mixture at
P
43 mNm
1
was observed, which may
be associated with a change in the miscibility of the lipids in the
monolayer. After this plateau, the DPPC:PIP
2
mixed monolayer iso-
therm was shifted to even lower molecular areas than those of
pure DPPC. The small values of C
s
1
at larger areas per molecule cor-
responding to both monolayers were rationalized by the existence
of a high compressibility LE region, whilst higher values of C
s
1
(130 mNm
1
for DPPC and 100 mNm
1
for DPPC:PIP
2
monolayers
at smaller areas per molecule) correspond to the LC phase. The
introduction of PIP
2
to the monolayer lead to a reduction in the
elasticity by a factor of 2.5, in the surface pressure range 30–
50 mN
.
m
1
, which corresponds to the LC DPPC phase.
To summarize, two observations can be made from the
P
-A iso-
therms: firstly, as previously reported for DPPC:PIP
2
mixed mono-
layers [14],the presence of PIP
2
does not drastically influence the
DPPC isotherm. This low impact of PIPs on model membranes
has been also observed in bilayers [11,21]. Secondly, in buffer with
Mg
2+
ions, the values of C
s
1
corresponding to the LC phase for
DPPC:PIP
2
mixed monolayers are much lower in comparison with
the DPPC monolayer [36]. However, in both systems, the values
of C
s
1
are remarkably smaller in comparison to those measured
for DPPC monolayers in water [33]. This effect can be attributed
to the presence of divalent Mg
2+
cations that decrease the lipid
packing density in mono- and bilayers [36,38–39]. Furthermore,
it is worth mentioning that the steric hindrance associated with
the size of the PIP
2
headgroup may hinder partially the maximum
lipid packing in DPPC:PIP
2
monolayers, and hence contributes to
the reduction of C
s
1
with respect to DPPC monolayers, indepen-
dently of the ionic content of the subphase.
3.2. In-plane organization of DPPC:PIP
2
monolayer investigated
through Brewster angle microscopy
A series of BAM images taken during the lateral compression of
pure DPPC and DPPC:PIP
2
mixed monolayers are reported in Fig. 2
E-J, respectively. Different lateral organization of the lipids at the
interface was observed depending on the specific composition
and compression state, i.e., lipid area per molecule. Indeed, BAM
is sensitive to the phase behaviour within the plane of the mono-
layer and it does not require the use of fluorescent dyes that could
introduce artefacts to the observation of lipid miscibility. Particu-
larly, the orientational order related to lipid condensed phases
results in modulations of the polarization state of the reflected
light [40–41]. As a consequence, dark areas in the BAM images
are attributed to fluid regions while more condensed phases are
seen as bright micro-domains.
At
P
=15mNm
1
, bean-shaped domains are visible for DPPC
monolayer in Mg
2+
ions containing buffer (Fig. 2 E), which appears
less condensed than DPPC monolayer spread on pure water (Fig-
ure S2). This morphological difference is likely due to the presence
of divalent cations (here, Mg
2+
) in the subphase, which have been
shown to increase lateral compressibility as well as permeability of
the monolayer [36,38–39]. BAM images became brighter at
increasing
P
indicating a reduction of the area occupied by the
fluid phase. At
P
=30mNm
1
, it is still possible to observe con-
densed domains in the DPPC monolayer that are coalescing as
the lateral packing is increasing due to compression (Fig. 2 F). At
very high surface pressures (
P
=41mNm
1
) the emergence of a
condensed phase for DPPC characterized by a rather homogenous
film is observed (Fig. 2 G). In the presence of pure water, DPPC
monolayer is in condensed state at lower pressures as it can be
seen in Figure S2.
In the case of DPPC:PIP
2
mixed monolayer, similar domains as
for DPPC monolayers were found at
P
=15mNm
1
(Fig. 2 H). In
particular, the dark areas in the image were assigned to a fluid
phase that includes both DPPC and PIP
2
molecules while the bright,
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
788
large domains to ordered DPPC molecules. In addition, bright, small
domains (size < 30
l
m
2
, see Fig. 3 B) also appeared in the image.
Interestingly, in DPPC:PIP
2
monolayers at
P
=30mNm
1
(Fig. 2
I), condensed domains are also clearly visible but strikingly coexist
with small bright spots, uniformly dispersed within the LE region
in a larger number with respect to
P
=15 mNm
1
(Fig. 2 H). We
attributed those small bright spots to PIP
2
clusters, which have
been already reported in the literature [4–5]. This aggregation of
PIP
2
molecules within the mixed monolayer may be ascribed to
the presence of Mg
2+
ions in the subphase, which bridge different
PIP
2
molecules together, thus excluding other lipids [42]. The small
size of these PIP
2
clusters in relation to DPPC domains is the result
of diffusion limitations [7]. Therefore, at
P
=30mNm
1
(Fig. 2 I)it
was possible to identify up to three different coexisting phases: (1)
Fig. 2. A Surface pressure - area isotherms of DPPC (black line), PIP
2
(red line) and DPPC:PIP
2
(violet line) monolayers. Compressional elastic modulus profiles of DPPC (black
circles), PIP
2
(red triangles) and DPPC:PIP
2
(violet circles) monolayers plotted versus B
P
and Carea per molecule. Dpanel showing the excess area per molecule (black
squares) and the Gibbs energy of mixing (dark blue triangles) calculated at the three pressures for the mixed monolayer. BAM image of DPPC monolayer in HKM buffer at E
15 mN
.
m
1
,F30 mN
.
m
1
and G41 mN
.
m
1
. BAM image of DPPC:PIP
2
mixed monolayer in HKM buffer at H15 mN
.
m
1
,I30 mN
.
m
1
and J41 mN
.
m
1
. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
789
a fluid phase region containing both DPPC and PIP
2
, (2) large DPPC
condensed domains, and (3) small bright PIP
2
clusters. Charge
shielding and cluster formation did not prevent the formation of
condensed DPPC domains that fuse at higher pressures. However,
these effects force a random orientation of the molecules at the
interface, yielding more disorganized monolayers. Finally, at
P
=41mNm
1
(Fig. 2 J), for which the
P
-A isotherm of DPPC:
PIP
2
monolayer showed a pseudo-plateau, fusion of the condensed
DPPC domains occurred, forming a honeycomb structure in which
the fraction of area occupied by the LE phase decreased dramati-
cally. It is also interesting to note that the size of PIP
2
domains
increased with the pressure, from 30 to 41 mNm
1
(Fig. 3 Aand
B). Besides, the position of PIP
2
clusters is clearly in the darker
(more fluid) zone at 30 mNm
1
, while at 41 mNm
1
PIP
2
domains
reorganised themselves at the interface between fused condensed
DPPC domains (medium bright regions) and more liquid phases
(dark regions). At high surface pressures (30 and 41 mNm
1
),
the phase separation between DPPC and PIP
2
, may play an impor-
tant role in PIP
2
function, as PIP
2
clustering directly increases its
local concentration, thereby influencing its interaction with other
molecules, like peripheral membrane proteins [7,10]. The cluster
ability of PIP
2
was also reported by Braunger et al. [43] in binary
model membranes containing also 1-palmitoyl-2-oleoyl-sn-gly
cero-3-phosphocholine (POPC). Their results suggested that PIP
2
cluster formation can be mediated by divalent cations, but is also
strongly influenced by hydrogen bond formation between the inos-
itol rings.
Moreover, BAM images were quantitatively analysed to gain
further insight into the evolution of DPPC micro-domains and
PIP
2
clusters with the increase in surface pressure, and, therefore,
the decrease in area per molecule. The domain size distribution,
in terms of the relative area with respect to the total area, of both
DPPC and PIP
2
domains was calculated for different surface pres-
sures and plotted in Fig. 3 A. The characteristic size of each popu-
lation is reported in Fig. 3 B, together with the corresponding
histograms fitted to a normal Gaussian distribution (Fig. 3 C,D
and E). On one side, the number of DPPC condensed domains
remains relatively constant, 25% of the total area (represented
as orange area in Fig. 3 Aand B) until a pressure of
P
35 mNm
1
is reached. Above that pressure (cyan area in area
in Fig. 3 Aand B), LC domains start to coalesce and the LE phase
(represented as blue triangle in Fig. 3 A,Band E) reached a final
distribution of 50% of the total area. The fraction of area corre-
sponding to the PIP
2
clusters increases with pressure, alongside
with their average size (Fig. 3). In this case, the clusters may be
formed by a nucleation/growth mechanism that is characterized
by the nucleation of novel clusters (visible an increase in the area
fraction with pressure) and the growth of the ancient ones already
formed. Interestingly, a reduction in DPPC domains size was
observed from 15 to 30 mNm
1
. We attributed this result to an
enhanced packing of the DPPC molecules within the domains, with
their orientation being more perpendicular to the plane of the
monolayer.
Fig. 3. Quantitative analysis of BAM images. DPPC domains are depicted as black squares, PIP
2
domains are represented as red circles and finally liquid expanded areas as blue
triangles. AArea fraction occupied by each domain versus surface pressure. BDomain size versus surface pressure. Histograms related to domain size fitted to a normal
Gaussian distribution at C15 mN
.
m
1
,D30 mN
.
m
1
and E41 mN
.
m
1
BAM images. Insets in C,Dand Ecorresponds to the 2D-FFT power spectra. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
790
Finally, to get insights into the lipid miscibility, the excess in
area per molecule (A
exc
) as well as the total Gibbs energy of mixing
(
D
G
mix
) were calculated (Equations S2 and S3) and plotted as
function of the surface pressure in Fig. 2 D. The resultant negative
values indicate the presence of attractive interactions between the
molecules, which enhance the cohesion between lipid molecules
within the mixed monolayer [44]. However, it is interesting to note
that, although still negative, the values peak at
P
=30mNm
1
lies
between the ones of 15 and 41 mNm
1
. This can be due to steric
effects, triggered by the insertion of PIP
2
molecules in DPPC
domains. The more condensed LC domains tend to exclude PIP
2
molecules, which in turn, form clusters in the LE phase of the
monolayer due to the presence of Mg
2+
ions. Finally, at
P
=41m
Nm
1
, the lower
D
G
mix
suggests a better mixing between lipids.
Indeed, BAM images clearly show that PIP
2
domains are not only
confined to the LE phase but are in close contact with LC DPPC
domains.
3.3. Out-of-plane structure of DPPC:PIP
2
mixed monolayers and
determination of the tilt angles by neutron reflectometry
NR allowed the determination of both the out-of-plane struc-
ture and the exact composition of the lipid monolayer. Indeed,
the experimental elucidation of the DPPC:PIP
2
monolayer composi-
tion was necessary to accurately determine the molecular volumes
and scattering length densities, SLD, values of the DPPC:PIP
2
mix-
ture. Thus, experimental data at a limited momentum transfer
range (0.01 Å
1
<Q
Z
< 0.03 Å
1
) were collected and analysed by
a one-layer model that is only sensitive to the composition of the
monolayer [45–46] (Fig. 1 Cand Supplementary Methods).By
using two different isotopic contrasts, hydrogenous DPPC (h-
DPPC):PIP
2
and chain-deuterated DPPC (d62-DPPC):PIP
2
in HKM-
buffered air contrast matched water (ACMW), the relative amount
of DPPC and PIP
2
at three surface pressures (15, 30 and 41 mNm
1
)
was determined, yielding values very close to the nominal lipid
composition (Table S2). From these molar compositions, and using
the values shown in Table S1, the values of molecular volume and
SLD for both h-DPPC:PIP
2
and d62-DPPC:PIP
2
mixtures at the three
pressures were calculated. These values, shown in Table S3, were
then included in the model of the experimental data at the fully
accessible Q
z
-range, to obtain information relative to the mono-
layer out-of-plane structure. In this case, four different isotopic
contrasts were exploited, employing both hydrogenous and
deuterated tails phospholipid. Thus, by spreading monolayers of
h-DPPC:PIP
2
and d62-DPPC:PIP
2
onto both HKM-buffered ACMW
and D
2
O subphases (see Figure S3 for experimental curves), it
was possible to define a two-layer model to fit the data, rationally
divided into polar headgroups facing the bulk phase and aliphatic
tails in contact with air (Fig. 1 Cand Fig. 4). The parameters opti-
mised during the fitting process (Table S4) were: thickness of both
headgroups and tails (t
heads
and t
tails
, respectively), water volume
fraction in the headgroups layer (f
w
) and roughness (r) of the three
interfaces (air/tails layer, tails layer/headgroups layer, headgroups
layer/subphase), which were constrained to be equal [33,36]. The
values of roughness obtained were compatible with that corre-
sponding to water capillary waves ()[47–49] and did not sig-
nificantly vary in the range of surface pressures studied. Besides,
each of the models used to fit the data ensure the same area per
molecule of tails and headgroups, whose values were consistent
with the ones obtained from isotherm and ellipsometry
(Table S5 and Figure S7). The optimised value of f
w
was in perfect
agreement with the value calculated according to Equation S7.
This finding further assures the goodness of our two-layers models.
The use of four different isotopic contrasts has allowed to opti-
mize simultaneously the different structural parameters (illus-
trated in Fig. 1 C) reported in Fig. 5 A, B, which clearly depend
on the monolayer compression. As expected, the increase in sur-
face pressure produced an increase in the thickness of the aliphatic
tails as shown in Fig. 5 A. This can be rationalized because further
compression diminishes the area available per lipid molecule,
yielding an increase of the tilt angle of the hydrophobic tails, as
already reported in literature [14]. Here, the tilt angle (/) of the
tails with respect to the membrane normal, calculated as /
¼cos
1
t
tails
=t
pal

with t
pal
the thickness of the palmitoyl chain
(19.5 Å [50], resulted in /=52°,44°and 35°for
P
= 15, 30 and
41 mN
.
m
1
, respectively (plotted against the corresponding A in
Fig. 5 F). Both values of /and A are larger with respect to the ones
of PIP
2
containing monolayer in the absence of divalent salts (i.e.,in
purely HEPES-NaCl-EDTA buffer) [14] confirming thus a decrease
in the packing density of the monolayers in the presence of diva-
lent salts, as already observed in purely DPPC monolayers [36].
Despite previous NR studies on DPPC monolayers report a con-
stant value of the headgroups thickness at different surface pres-
sures [33,51], here the values of t
heads
obtained experience an
increase with
P
(see Fig. 5 A), attributed to a change in PIP
2
head-
group tilt angle. Besides, the increase in f
w
observed (Fig. 5 B) can
be rationalized by the change of the headgroup tilt angle. Particu-
larly, the systematic increase in both t
heads
and f
w
were assumed to
be determined by the change in the orientation of the inositol ring,
which was further calculated below. Although it was already
Fig. 4. Data derived from fitting of reflectivity profiles plotted in Figure S4. The panels show the volume fraction profiles normal to the interface underlying the distribution of
tails (black), headgroups (olive blue) and water (cyan), at the surface pressure value of B15 mN
.
m
1
,C30 mN
.
m
1
and D41 mN
.
m
1
. PIP
2
volume fraction, in both tails- and
headgroups layers, is shown as red lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
791
Fig. 5. A Thickness of tails (green triangles) and headgroups (blue circles) plotted versus surface pressure, together with total thickness of the monolayer (black squares). B
Volume fraction of water in the headgroups layer plotted versus surface pressure. CPlot showing the thickness increment of the headgroups layer obtained from the fit versus
surface pressure. The linear dependence is shown as a red line. DThe calculated values of tilt angle (
s
) versus surface pressure are shown as black squares. The fit performed
using a cos
1
function (Equation (1)) is shown as a red line. The interpolated point (35 mN
.
m
1
;40°)[20–21] is shown in blue. ESketches representing mixed DPPC:PIP
2
monolayer at the three pressures. The inositol-biphosphate (IP
2
) ring is depicted in red. The normal to the monolayer (dashed grey line) and the vector connecting C1 and C4
of the inositol-biphosphate (IP
2
) ring (v
14
, dashed green line) are shown. The tilt angle (
s
), which is calculated between the normal and v
14
, is shown and depicted as light
brown area. FComparison between out-of-plane structure and in-plane organization of DPPC:PIP
2
mixed monolayers at the three surface pressures. The central panel reports
the values of PIP
2
inositol ring tilt angle (
s
) and tails tilt angle (/) versus area per molecule. Left panel shows the monolayer structure at a low area per molecule value as well
as the graphical definition of
s
and /. Right panel reports a sketch of PIP
2
cluster in the mixed monolayer at larger areas. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
792
demonstrated by NR that the inositol-triphosphate ring assumes a
preferred orientation close to the surrounding lipid headgroups in
bilayers [11], the tilt angle of the ring has been never experimen-
tally determined, to the best of our knowledge. Molecular dynamic
(MD) simulations on lipid bilayers [3,20–21] suggested that PIP
2
adopts a preferred orientation of 40°with respect to the mem-
brane normal. Noteworthy, these simulations demonstrated that
the solvent-accessible surface area of the PIP
2
headgroup increases
as the tilt angle decreases, since fewer water molecules can be pre-
sent in the solvation shell of the inositol ring when oriented closer
to the membrane. Our experimental results from NR gave similar
outcomes concerning the PIP
2
headgroup hydration (Table S4
and Fig. 5 B): as surface pressure increases, although the head-
group layer thickens as the tilt angle decreases, its water content
increases.
To calculate the PIP
2
tilt angle, denoted as
s
, the inositol-(1,4,5)
triphosphate ring’s average size 7Å(t
IP
2
) was estimated from its
atomic structure (as found in the crystal structure PDB: 1HFA [12]),
measuring the distances between the oxygen atom bound to C1
and P1 and the oxygen atoms bound to P4 and P5 (Fig. 1 B). The
phosphate P1 was considered to be at the same position (along
z) than the phosphate of DPPC, as reported by Wu et al. [21].
Assuming this model, at
P
=15mN
.
m
1
the inositol-biphosphate
ring would lie parallel to the monolayer (
s
=90°), as the head-
groups thickness obtained (t
heads
= 7 Å) is similar to the value for
a pure DPPC monolayer [52–53].
The linear dependence in the headgroup thickness with
P
,
defined as
D
t
heads
¼t
heads
P
ðÞt
heads
ð15mN
:
m
1
Þ, and reported in
Fig. 5 C, could be attributed exclusively to the change in the molec-
ular orientation of the inositol-biphosphate ring. Thus, a protrusion
of PIP
2
headgroup happens. It is important to underline that similar
protrusions were already observed by MD simulation for DPPC:PIP
2
bilayers [3]. Considering t
IP
2
the inositol-biphosphate ring length
and
D
t
heads
the projection of t
IP
2
onto the z-axis, normal to the
monolayer (Figure S4), the PIP
2
tilt angle
s
can be calculated as
s
¼cos
1
D
t
heads
=t
IP
2

. Its dependence on the monolayer compres-
sion and, therefore, on the surface pressure, could be empirically
described by
s
¼cos
1
a
P
þb
t
IP
2

;ð1Þ
where, aand bare constants defined by the linear relationship
between
D
t
heads
and
P
. The trajectory described by
s
at varying
P
due to monolayer compression, shown in Fig. 5 D, is in excellent
agreement with the calculated values of
s
obtained from previous
MD simulations [3,20–21]. Indeed, a bilayer with an A of 50 Å
2
and characterized by a hypothetical surface pressure of 35 mN
.
m
1
(extrapolated from the isotherm), yields a calculated PIP
2
tilt angle
of
s
40°(blue circle in Fig. 5 DA). This value of area per molecule
is compatible with the one of a DPPC bilayer at 20 °C (gel phase)
[54].
To summarize, the aliphatic tails (/) and inositol ring (
s
) tilt
angles calculated were plotted against A as shown in Fig. 5 F. The
values of A were also calculated by NR and ellipsometry (Supple-
mentary Methods and Figure S7). As the lipid leaflet was com-
pressed, lipids gain in compaction and thus reduce their surface
area. This is reflected not only in the decrease of /, but also in
the re-orientation of PIP
2
headgroups towards the bulk phase,
illustrated by the decrease of
s
with A. This tendency can be corre-
lated with the in-plane structure observed in the BAM images, in
which there is an increase in PIP
2
clustering at larger areas per
molecule possibly due to the re-organization of the PIP
2
head-
groups occupying lower volume and favouring less steric hin-
drance between them. The collapse of the DPPC:PIP
2
monolayer
was observed slightly above
P
=43 mN
.
m
1
as shown Fig. 2A.It
is usually characterized by the loss of lipids from the interface,
and/or the buckling of the monolayer yielding to defects in the film
and/or folded structures [57]. In any case, the existence of those
structures might induce errors in the calculation of the PIP
2
ring tilt
angle from the fitting of NR experimental data and, therefore, only
surface pressures in the LC phase were studied by NR.
4. Conclusions
PIP
2
, the most abundant inositol phospholipid in the inner leaf-
let of the plasma membrane, is responsible for the interaction and
recruitment of different proteins involved in key biological pro-
cesses (e.g., endocytosis, ion channel activation and secondary
messenger production [1–3]). Its phosphorylated inositol ring is
responsible for specific protein binding via electrostatic interac-
tions [55–56]. Hence, it is crucial to investigate its orientation with
respect to the plane of the membrane. However, this parameter
had only been inferred from molecular dynamic simulations of
model membranes [20–21]. Here, we designed a simple, in vitro
lipid model system, in the form of Langmuir monolayer, composed
of PIP
2
and DPPC. By using an experimental approach based on dif-
ferent surface sensitive techniques, we aimed to describe and
rationalize the relation between the in-plane organization of PIP
2
molecules and the molecular orientation of their inositol ring, in
terms of tilt angle, in monolayers as function of the interfacial
lipids packing density, i.e., different areas per molecule. This has
allowed the study of the evolution of PIP
2
clusters in our planar
model system, when divalent cations are present in bulk at differ-
ent degree of lateral packing of the monolayer compression. In par-
ticular, this work has been focused on establishing a relation
between PIP
2
domain formation and the monolayer out-of-plane
structure determined by neutron reflectometry. PIP
2
cluster forma-
tion depends not only on the presence of Mg
2+
ions, which bridge
different PIP
2
molecules together [42], but also on the surface pres-
sure, which influences the out-of-plane structure of the lipids.
Moreover, neutron reflectometry has allowed the determination
of the inositol-phosphate ring orientation, in terms of its tilt angle.
We have observed, for the first time, that when the inositol-
biphosphate ring moves away from the membrane, the formation
of clusters is favoured, and larger PIP
2
islands can form. This beha-
viour is also identified from the quantitative analysis of the BAM
images. Indeed, PIP
2
clusters (with an area > 1%) form from
P
=
30 mN
.
m
1
and their number increases until a plateau at
P
=35
mN
.
m
1
is reached. Therefore, our results clearly show the depen-
dence between the orientation of the inositol-biphosphate ring and
the PIP
2
cluster formation. Moreover, an empirical equation has
been proposed to determine the trajectory of the PIP
2
headgroups
tilt angle with the surface pressure and the level of monolayer
compression. The orientation of PIP
2
headgroup can thus be esti-
mated at different molecular areas and experimental conditions.
The investigation of the lipid in-plane organization, by means of
BAM, showed the formation of PIP
2
cluster in presence of divalent
cations, which have been already reported in the literature [4–
5,42]. However, in this work we were able to correlate cluster for-
mation with the out-of-plane structure of the monolayer, i.e., tilt-
ing of both acyl chains and PIP
2
inositol-biphosphate ring.
Moreover, our experimental outcomes agree with previous molec-
ular dynamic simulations that calculated the preferred inositol-
biphosphate ring orientation in PIP
2
-containing lipid bilayers
[3,20–21] (40°, with respect to the membrane normal). Indeed,
by using Equation (1), this value leads to a surface pressure of
35 mN
.
m
1
, corresponding to the typical bilayer area per molecule
phase [54]. To the best of our knowledge, this is the first time that
the orientation of inositol-phosphate ring has been determined
experimentally.
A. Santamaria, J. Carrascosa-Tejedor, E. Guzmán et al. Journal of Colloid and Interface Science 629 (2023) 785–795
793
Finally, we consider that our work will help to a better under-
standing of both PIP
2
-protein interaction and PIP
2
role in mem-
brane curvature. Indeed, the PIP
2
headgroup variation of
orientation found here might facilitate changes in membrane cur-
vature. Particularly, we explored the limiting case at a very low
surface area characterized by an orthogonal position of the inositol
ring with respect to the plane of the membrane that turns into an
almost parallel orientation at larger areas. This gradual PIP
2
head-
group protrusion in the bulk phase observed at decreasing surface
areas might have a direct influence on membrane curvature mod-
ulating the binding of different peripherical membrane proteins.
CRediT authorship contribution statement
Andreas Santamaria: Methodology, Investigation, Formal anal-
ysis, Data curation, Writing original draft, Writing review &
editing, Visualization, Software. Javier Carrascosa-Tejedor:
Methodology, Investigation, Formal analysis, Data curation, Writ-
ing review & editing, Visualization, Software. Eduardo Guzmán:
Writing review & editing, Visualization, Validation, Supervision.
Nathan R. Zaccai: Writing review & editing, Visualization, Vali-
dation, Supervision. Armando Maestro: Project administration,
Conceptualization, Methodology, Writing review & editing, Visu-
alization, Validation, Supervision, Funding acquisition, Resources.
Data availability
Data will be made available on request.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The authors thank the Institut Laue-Langevin for the allocation
of beamtime, the Partnership for Soft Condensed Matter (PSCM) for
the lab support and Giovanna Fragneto for the critical reading of
the manuscript. E.G. and A.M. acknowledge the financial support
from MICINN under grants PID2019-106557GB-C21 and
PID2021-129054NA-I00, respectively. A.M. also acknowledges the
financial support received from the IKUR Strategy under the collab-
oration agreement between Ikerbasque Foundation and Materials
Physics Center on behalf of the Department of Education of the
Basque Government.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcis.2022.09.095.
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