BBA - Biomembranes 1865 (2023) 184113

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BBA - Biomembranes
journal homepage: www.elsevier.com/locate/bbamem

Pro-inflammatory protein S100A9 alters membrane organization by

dispersing ordered domains
Rimgailė Tamulytė a, Evelina Jankaitytė a, Zigmantas Toleikis b, Vytautas Smirnovas b,
Marija Jankunec a, *
a
Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, Vilnius LT-10257, Lithuania
b
Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, Vilnius LT-10257, Lithuania

A R T I C L E I N F O A B S T R A C T

Keywords: Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates
Aggregation several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the
Atomic force microscopy pathogenesis of Alzheimer’s disease (AD). The number of studies on the impact of S100A9 in co-aggregation
S100A9
processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein
Lipid membrane remodeling
interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS),
Phase separation
Tethered lipid bilayers and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the
membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane
models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further
loss of the bilayer’s integrity. The most important result is that the initial adsorption and accumulation of apo-
form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and
disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid
bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain
total lipid extract). Understanding the function and interactions of these proteins in cellular environments might
expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.

1. Introduction
Inflammation is an organism’s natural defensive response to harmful
stimuli [1]. Lately, it has been named as a leading cause of Alzheimer’s
disease (AD) pathogenesis and as a factor promoting the progression and
severity of this illness [2,3]. Notably, a pro-inflammatory S100A9 pro­
tein has been a subject of high interest [4–7]. This calcium-binding
protein contains two helix-loop-helix (EF-hands) motifs and belongs to
the S100 protein family. The protein is permanently expressed in
monocytes, neutrophils, dendritic cells, and other cells as macrophages
upon activation [8–10]. Furthermore, S100A9 is highly expressed and
plays a prognostic role in various cancers, including lung [11], gastric
[12], and others [13,14]. The S100A9 proteins are calcium sensors
changing their conformation in response to calcium influx [15,16]; this
mediates interactions with membrane-associated molecular targets
[17,18]. Intracellular free Ca2+ concentration widely varies depending
on its location. Typically, in the mammalian brain, the concentrations
are maintained low, roughly 0.1–0.5 μM [19]. The extracellular free
calcium concentration typically ranges from 1.5 to 2.0 mM [20]. In the
presence of calcium ions, S100A9 changes the conformation to expose a
more hydrophobic surface to solvent [15], facilitating its translocation
into the extracellular space [21]. When the extracellular S100 proteins
have all calcium-binding sites occupied, the protein’s structure is sta­
bilized [15], and interactions with target proteins or membranes are
promoted [17]. Under the cell resting conditions, when the cytosolic
free calcium concentration is maintained at the nanomolar level, S100
proteins occupy a closed, relatively hydrophilic conformation [16].
The structural changes of S100 proteins upon binding calcium are
reflected in the affinity toward lipid bilayers. One example is S100D, of
which calcium-free form is associated with detergent dodecyl phos­
phocholine (DPC) micelles, but site-specific binding with individual DPC
molecules occurs when Ca2+ ions are bound [22]. Another study showed
that both forms of S100A12 protein interact with both zwitterionic and
negatively-charged membranes [23]. The interactions with ions and

* Corresponding author.
E-mail address: marija.jankunec@gmc.vu.lt (M. Jankunec).

https://doi.org/10.1016/j.bbamem.2022.184113
Received 31 May 2022; Received in revised form 6 December 2022; Accepted 16 December 2022
Available online 23 December 2022
0005-2736/© 2022 Elsevier B.V. All rights reserved.
R. Tamulytė et al.
liposomes might work as a molecular triggering mechanism for the
protein to adopt adequate conformation and to find its targets on the
membrane surface. Increasing evidence shows that protein interaction
with cell membranes plays a critical role in the accumulation and
growth of various amyloidogenic proteins [24,25]. The heterogeneous
aggregation promoted by protein crowding on the membrane surface
increases the local concentration of proteins and shapes the lipid
membrane [26]. Since S100A9 possesses amyloid-like properties [27],
the lipid bilayer’s closeness might affect the protein’s aggregation rates.
However, the data for this protein is lacking in the literature. One study
showed that the mature S100A9 aggregates were co-localized with
monosialotetrahexosylganglioside-1 (GM1) in neuroblastoma SH-SY-5Y
cells in vitro [28]. These data support the hypothesis that the binding
sites of S100A9 amyloids to the cell membrane occur at the raft domains.
These observations also suggest the critical role of GM1 in protein-
membrane association, most likely due to the clusters of sialic acid
moieties, producing negatively charged regions at the cell surface. In
another study, the formation of large aggregates of the S100A8/S100A9
complex upon incubation with the model phosphatidylcholine/choles­
terol lipid bilayer was observed [29]. This complex translocates into a
detergent-resistant lipid structure only after calcium activation of the
neutrophils. However, the S100A9/A8 membrane association is
cholesterol and sphingolipid-independent, not restricted to lipid rafts
[18].
This work aims to study the affinity of the apo-form (calcium-free) of
pro-inflammatory S100A9 protein toward lipid bilayers. The surface
sensitive-technique atomic force microscopy (AFM), dynamic light
scattering (DLS), and Thioflavin-T (ThT) fluorescence assay allowed us
to study the accumulation and aggregation of S100A9 protein and
morphological changes in the affected lipid bilayer surface. Two cell
membrane biomimetic platforms were used. Tethered bilayer lipid
membranes (tBLMs) were used as a model on a solid substrate, while
unilamellar liposomes represented the lipid bilayer in bulk solution. The
initial stage of protein-lipid interactions is influenced by electrostatic
interaction between hydrophilic parts of protein and the charge of the
headgroup of lipids. The fluidity of the lipid bilayer could be modulated
by including sterols and saturated and unsaturated backbone lipids.
Thus our chosen lipid membranes contain 60 mol% of unsaturated lipid
dioleoylphosphatidylcholine (DOPC). The other lipids with a negatively
charged headgroup (dioleoylphosphatidylserine, DOPS) and saturated
acyl chains (dipalmitoylphosphatidylcholine, DPPC), or cholesterol,
would cover the rest 40 mol%. Next, lipid bilayers composed of brain
total lipid extract combine both (fluidity and negative charge) properties
in one. Additionally, we used more complex lipid compositions to mimic
the neuronal membrane — DOPC/dioleoylphosphatidylethanolamine
(DOPE)/Chol/brain sphingomyelin (bSM)/GM1 [30]. Here, for the
latter composition, we expect the formation of lipid-raft-like domains
that are enriched with cholesterol, sphingomyelin, and, in particular,
GM1 [31].
2. Experimental
2.1. Materials
All lipids were purchased from Avanti Polar Lipids (Alabaster, USA),
including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dio­
leoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-dipalmi­
toyl-sn-glycero-3-phosphocholine (DPPC), GM1 ganglioside (brain,
ovine‑sodium salt), brain sphingomyelin (bSM, porcine), brain total
lipid extract (BTLE, porcine, composition by weight: PC (9.6 %), PE
(16.9 %), PS (10.6 %), PI (1.6 %), PA (2.8 %), unknown 58.7 %), 1,2-
dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). PBS buffer (pH
7.2) containing 130 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8
mM KH2PO4 (Sigma Aldrich, Hamburg, Germany). The saline solution
containing 0.1 M NaCl, 0.01 M NaH2PO4 (Roth, Frederikssund,
Denmark), pH 4.5. S100A9 protein (1.9 mg/ml, molecular weight is
2
BBA - Biomembranes 1865 (2023) 184113
~13.2 kDa) was dissolved in PBS buffer at pH 7.2, with all solutions
remaining on ice. No further actions were conducted before the analysis.
Lipid compositions used in this work: DOPC/Chol 60/40 mol% —
zwitterionic lamellar phase; DOPC/DOPS 60/40 mol% — negatively
charged anionic liquid-disordered (Ld) phase; DOPC/DPPC 60/40 mol%
— Ld/gel phase (diphasic system); neuronal membrane biomimetic,
lipid raft-like system — DOPC/DOPE/bSM/Chol/GM1 (44/20/30/5/1
mol%) and brain total lipid extract.
2.2. Methods
2.2.1. Preparation of multilamellar and unilamellar vesicles
Initial lipid mixtures were dissolved in chloroform and dried under
N2 for 40 min. After solvent traces removal and formation of thin lipid
film, lipids (the final concentration of 1 mM) were rehydrated: i) in
saline solution (pH 4.5) to obtain multilamellar vesicles for AFM ex­
periments; and ii) in PBS-buffer, subjected five times to freeze-thaw
cycles, ultrasonicated for 1 h and subsequently extruded 21 times
through 100 nm polycarbonate membranes with a mini-extruder
(Avanti Polar Lipids Inc., USA) to obtain unilamellar vesicles (LUVs).
The extrusion was done above the lipids’ melting temperature. The LUVs
size distribution was determined by dynamic light scattering using a
Malvern Zetasizer μV apparatus (Malvern Panalytical, UK). Z-average ±
SEM calculated based on size distribution per mass; n = 3, the number of
runs per sample is 4.
2.2.2. Laurdan spectroscopy
Prepared LUVs were mixed with Laurdan (dissolved in dimethyl
sulphoxide) to give a final probe-to-lipid ratio of 1/200. Samples were
pre-incubated 1 h before fluorescence measurements on ClarioStar Plus
(BMG Labtech, Germany) plate reader. Before measurements, liposome
suspensions were allowed to equilibrate for 10 min at each temperature.
Laurdan emission spectra were recorded from 400 to 600 nm using a
340 nm excitation wavelength. The excitation and emission band-pass
bandwidth was 10 nm. The generalized polarization (GP) quantifies
the Laurdan emission spectrum changes. In analogy with fluorescence
polarization:
I440 − I490
GP = , (1)
I440 + I490
where I440 and I490 are the fluorescence intensities registered at the
maximum emission wavelength in the ordered (440 nm) and disordered
(490 nm) phases [32]. All experiments were performed at least three
times with triplicate assays.
2.2.3. Formation of tethered lipid bilayer membranes (tBLMs)
Freshly cleaved mica (grade IV, SPI Supplies, USA) sheets with
annealed gold layers were prepared by magnetron sputtering using a
PVD75 system (Kurt J. Lesker Co., USA). The typical thickness of the
gold layer was 100 nm, while the adhesive Cr layer was 10 nm. Then,
modification with self-assembled monolayers (SAMs) via incubation in
the ethanolic solution of WC14/βME (20-tetradecyloxy-
3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol/β-mercaptoethanol,
in the molar ratio 35/65, the total thiol concentration 0.05 mM) for 12 h.
The details on the synthesis and properties of the self-assembled
monolayers used in this work are described elsewhere [33]. After in­
cubation, the samples were rinsed with ethanol, dried under a nitrogen
stream, and stored dry until further use. Tethered lipid bilayers (tBLMs)
were formed via incubation of multilamellar vesicles (0.1 M NaCl, 0.01
M NaH2PO4, pH 4.5) of various lipid compositions for 0.5 h on SAMs-
covered mica substrates [34]. The incubation was done above the
melting temperature of the lipids at 50 ◦ C. After tBLMs formation,
samples were thoroughly rinsed with lipid-free PBS buffer (pH 7.2).
Fig. S6 (see Supplementary Materials) shows a schematic view of tBLMs
constructs.
R. Tamulytė et al.
2.2.4. Expression and purification of recombinant S100A9 protein
The plasmid containing human S100A9 coding DNA (construct
described previously [35]) was chemically transformed into E. coli BL21
(DE3) one-star strain. A single colony from transformed cells was
selected and grown overnight in 100 ml LB medium supplemented with
100 mg/ml ampicillin at 37 ◦ C. The cells were grown until the culture
reached an optical density of 0.6 at 600 nm, and then 3 ml of this culture
was transferred to 300 ml of ZYM-5052 auto-inductive medium with
ampicillin (100 μg/ml) for protein expression. The culture was grown
overnight at 37 ◦ C, 220 RPM; cells were harvested by centrifugation at
5000 ×g for 15 min at 4 ◦ C and frozen at − 20 ◦ C.
Before the purification, the cells pellets were thawed and resus­
pended in 20 mM Tris buffer (pH 8.0) with 0.5 mM EDTA and 0.5 mM
DTT buffer. Homogenized and sonicated for 20 min (50 % amplitude,
cycles of the 30 s ON, 30 s OFF). The pellet was collected by centrifu­
gation for 25 min at 20,000 ×g, 4 ◦ C, and the supernatant was discarded.
The washed pellets containing S100A9 protein were dissolved in 8 M
urea solution and refolded at 4 ◦ C via dialysis against 20 mM Tris buffer,
containing 0.5 mM EDTA and 0.5 mM DTT, pH 8.0. The dialysis buffer
was changed 4 times during 16 h of refolding. After refolding, the pre­
cipitant was removed by centrifugation for 25 min (20,000 ×g, 4 ◦ C).
The supernatant was mixed with a weak anion exchange chromatog­
raphy resin (DEAE Sepharose Fast Flow, GE Healthcare) and loaded on a
column. S100A9 was eluted when a linear gradient reached 0.25 mM
NaCl concentration. The fractions containing S100A9 protein were
concentrated to 7 mg/ml using Amicon®Ultra-15 centrifugal filters
(Millipore) with 10 kDa molecular weight cut-off (MWCO) membranes
and purified further by the size exclusion chromatography (Superdex 75,
GE Healthcare) in PBS. The fractions with S100A9 protein were ali­
quoted to tubes by 0.5 ml and stored at − 80 ◦ C freezer.
2.2.5. S100A9 aggregation and ThT fluorescence assay
Fluorescent dye Thioflavin-T (ThT) (Sigma-Aldrich, Germany) binds
to the β-sheet containing amyloid structures [36]; this leads to an in­
crease in its fluorescence and thus enables the kinetics of amyloid for­
mation to be followed and quantified. ThT was dissolved in PBS buffer
and filtered through a 0.2 μm syringe filter. Then the concentration of
ThT was determined using an extinction coefficient of 36 mM− 1 cm− 1 at
412 nm. The aggregation of 50 μM S100A9 in PBS (pH 7.2, containing
100 μM ThT (protein/ThT ratio of 1/2)) in the absence or presence of the
lipid membrane of various compositions (LUVs concentration was 500
μM) was monitored by ThT fluorescence in 96-well microplate (Corning
Inc., USA) on a reader ClarioStar Plus (BMG Labtech, Germany). The
volume of the reaction well was 100 μl. The ThT fluorescence intensity
of each sample was recorded every 5 min with 440 nm excitation and
490 nm emission filters set at 37 ◦ C. The excitation and emission band-
pass bandwidth was 10 nm. The sample was constantly shaking at 400
rpm between measurements.
2.2.6. AFM Imaging
For AFM sample preparation in the air, the S100A9 stock solution
was diluted to 100 nM or 5 μM with PBS (pH 7.2), and 20 μl of the so­
lution was placed onto a freshly cleaved mica (grade 4, SPI Supplies,
USA) surface. After 2 min incubation, the sample was rinsed several
times with MilliQ-H2O and dried with a nitrogen airflow. AFM images
were obtained using Dimension Icon (Bruker, USA) AFM, operating in
tapping mode. For imaging in the air, tapping mode silicon nitride
probes FESP (Bruker, USA) with a nominal spring constant of 2.8 N/m
and resonance frequency of 75 kHz were used, nominal tip radius of 8
nm. Height images were collected (512 × 512 pixels) at scan speeds of
0.6–0.8 kHz.
For imaging of tBLMs, tapping-mode or PeakForce QNM were used in
PBS buffer at room temperature. AFM imaging in the fluid was per­
formed using triangular silicon nitride probes SNL-C (Bruker, USA) with
a nominal spring constant of 0.24 N/m and resonance frequency of 56
kHz in air. Height and phase images were collected (512 × 512 pixels) at
3
BBA - Biomembranes 1865 (2023) 184113
scan speeds of 0.3–0.5 kHz. After pre-visualization, tBLMs were exposed
to S100A9 solution in PBS (pH 7.2) (the final protein concentration was
5 μM; 37 μl of protein stock solution was added into AFM liquid cell
filled with 2 ml buffer) and imaged immediately. As the injection is done
locally near the scanning area, the local concentration of peptides may
vary due to uneven distribution in the fluid cell. The lag time between
adding the protein to the tBLM and starting the first image acquisition is
at least 10 min. Membrane imaging was conducted at ambient room
temperature in a liquid cell in PBS, pH 7.2, with hydration of the
membrane maintained throughout imaging. Successive AFM images
were recorded for several hours at the exact area unless otherwise
stated. Tethered lipid bilayers (tBLMs) formed from respective multi­
lamellar vesicles on the SAM-covered gold-coated mica sheets are me­
chanically stable over a long period. The average image acquisition time
is 20 min. Thus the first complete AFM image is recorded 30 min after
protein injection. All AFM measurements were performed at room
temperature (22 ◦ C). First, we imaged a 5 μm × 5 μm area for at least an
hour to verify the stability of the prepared bilayer and that no defects
were present within the deposited membrane. Each time, the lipid
bilayer formation was confirmed by a ramp to obtain force curves.
The image’s surface roughness is expressed by the arithmetic average
(Image Ra) value. Image Ra was assigned to each represented AFM image
in this work to show changes in a particular area of interest. Image Ra is
an average of the absolute values of the surface height deviations
measured from the mean plane:
1 ∑N ⃒⃒ ⃒⃒
Ra = Zj . (2)
N j=1
Multiple images of different scanning areas were taken for each
sample. The images were flattened and analyzed using NanoScope
Analysis 1.9 (Bruker, USA) and WSxM 4.0 [37] software.
2.3. Statistical analysis
Statistical analyses and the Gauss distribution were performed using
GraphPad Prism version 8.4.3 for Windows (GraphPad Software, USA),
and data were analyzed using a t-test. Significance was ascribed at P <
0.05. The data of different parameters were represented as the means ±
standard deviation if stated otherwise.
3. Results and discussion
This study aimed to assess calcium-unrelated effects and eliminate
factors that impact the properties of the lipid bilayer to highlight only
protein-driven interactions. Calcium ions interact with lipids’ head­
groups, causing dehydration of the phosphate [38], changing the
thickness [39], and increasing the ordering [40] of phospholipid bi­
layers. It is known that the binding of Ca2+ ions with negatively charged
lipids (ex., phosphatidylserine) and hydration effect create local changes
in the charge via the formation of negatively and positively charged
patches [41] or induce phase separation [42]. All these factors could
impact the lipid bilayer’s permeability or cause deformation not
detectable by atomic force microscopy (AFM) or fluorescence tech­
niques. These local deviations in the integrity of the lipid bilayer could
be primary targets of protein binding.
3.1. The morphology of initial protein preparation
Characterization of freshly thawed S100A9 protein (114 amino
acids) was assessed by AFM, allowing us to confirm the monomeric/
oligomeric state of the protein at the starting point of the protein-
membrane interaction experiment. Fig. 1a shows an AFM topography
image of the freshly thawed S100A9 protein solution (5 μM) adsorbed on
freshly cleaved mica and imaged in the air. The height and diameter
distributions (Fig. 1b and c panels) show the presence of particles with a
mixed size distribution, preferentially observed with sizes ranging from
R. Tamulytė et al.
Fig. 1. The morphology of the freshly dissolved S100A9 deposited on freshly cleaved mica. (a) AFM topography image visualized in air. The protein concentration is
5 μM. The scan size is 1 μm × 1 μm. Gauss distributions for height (b) and diameter (c) are 0.89 ± 0.23 nm (n = 649) and 25.4 ± 3.7 nm (n = 681), respectively. The
estimated protein’s hydrodynamic radius in solution by DLS (d).
15 to 45 nm in diameter and 0.6–1.5 nm in height. The height and
diameter Gauss distributions (GraphPad Software, Inc.) are 0.89 ± 0.23
nm and 25.4 ± 3.7 nm, respectively. Fig. 1S shows a close look at in­
dividual proteins and their profiles. Here we have used a geometrical
method to estimate the protein’s volume. The volume was calculated by
considering the protein as a spherical cap. The height and the full width
at half maximum were measured via cross-section analysis [43]. The
dimensions of a single S100A9 monomer structure were approximately
7.90–13.62 nm wide and 0.56–0.71 nm high, giving an experimental
molecular volume for the monomer as 26–31 nm3. These results agree
favorably with the calculated molecular volume of the S100A9 mono­
mer of 25 nm3 and are in line with previously reported values [44]. The
tip convolution was not taken into account. Based on the assumption
that the S100A9 monomer’s height is 0.50–0.75 nm, we conclude that
24.8 % of observed protein particles are monomers.
AFM in the air visualized the morphology of adsorbed molecules;
however, due to adsorption and surface-induced aggregation, molecules
tend to deform. Therefore, dynamic light scattering (DLS) data repre­
sents average size distribution in bulk. The estimated hydrodynamic
radius of S100A9 protein is 2.4 ± 0.5 nm (see Table 1, Fig. 1d), and
polydispersity is 0.22. The obtained particle size is close to the theo­
retical value of 2.1 nm for folded (globular) protein based on its mo­
lecular weight [45]. We conclude that derivatives of low
oligomerization dominate the initial protein stock solution. These data
allow us to understand what structures might populate the membrane’s
surface. All protein-lipid membrane interaction measurements were
performed on a rough gold-coated surface. Therefore, very small olig­
omers may be overlooked.
3.2. Determination of membrane lipid packing
Disordered proteins can undergo structural rearrangements upon
Table 1
DLS data of liposomes incubated with S100A9 protein for 24 h, n = 3.
Time Composition Zaverage ± SEM (nm)
0h S100A9 alone 2.4 ± 0.4
BTLE 127.4 ± 30.2
DOPC/Chol 133.8 ± 21.0
24 h, 37 ◦ C S100A9 alone 43.8 ± 4.6
S100A9 + BTLE 137.4 ± 11.8
S100A9 + DOPC/Chol 135.6 ± 12.6
4
BBA - Biomembranes 1865 (2023) 184113
binding with cell membranes. Moreover, the lipid bilayer’s biophysical
characteristics impact protein accumulation and its ability to penetrate
the lipid bilayer. Three of the five lipid compositions used in this work
are well-characterized membrane models: i) at room temperature, the
binary lipid system of zwitterionic DOPC and cholesterol (60/40 mol%)
based on X-ray [46] and NMR [47] data is in a liquid-ordered phase; ii)
the zwitterionic/anionic DOPC/DOPS (60/40 mol%) system consists of
two unsaturated lipids of the same acyl chain, with phase transition
temperatures − 17 and − 11 ◦ C [48], respectively, forms liquid-
disordered (Ld) phase; and iii) DOPC/DPPC (60/40 mol%) liposomes,
whereas DOPC is unsaturated and DPPC is a saturated lipid, form phase-
separated regions of Ld and gel (Lβ) phases [49]. Nevertheless, there is a
lack of data for complex BTLE and five-component lipid bilayers. To
address these issues, we used the Laurdan spectra to identify the mem­
brane lipid packing. Laurdan fluorescence emission spectra and gener­
alized polarization (GP) values for all well-characterized compositions
are available in Supplementary Materials Fig. S7.
Fig. 2 displays the evaluation of the lipid packing of BTLE and five-
component (DOPC/DOPE/Chol/bSM/GM1) large unilamellar vesicles
(LUVs) monitored at 22, 37, and 60 ◦ C by the Laurdan fluorescence
assay. After excitation at 390 nm, the values for the solid and fluid
phases were recorded at 440 nm and 490 nm, respectively. The wave­
lengths of interest are marked in Fig. 2a with dash lines. To compare the
lipid packing/order of these bilayers, we measured the GP values ac­
cording to Eq. 1. The measured mean GP value for BTLE composition at
room temperature is 0.37 ± 0.01 (n = 4), which decreases to 0.32 ±
0.02 at 37 ◦ C and 0.18 ± 0.01 when liposomes are heated up to 60 ◦ C.
The registered values are the highest among studied compositions,
indicating that prepared BTLE liposomes possibly are in a solid state.
The GP value for the five-component system is 0.10 ± 0.02 at room
temperature, which slightly decreases to 0.04 ± 0.01 and − 0.07 ± 0.02
at 37 and 60 ◦ C, respectively. The obtained GP value for the five-
component liposomes is higher than for the homogeneous Ld phase
(DOPC/DOPS) but lower than measured for coexisting Ld-Lβ system
(DOPC/DPPC).
The reduction in lipid packing with temperature increase indicates
the transition from a solid to a fluid state. However, the GP is an average
value, and the coexistence of different phases might be misinterpreted
[50]. Bagatolli et al. [51] showed that Laurdan distribution in the lipid
membranes is heterogeneous, whereas dye molecules partition prefer­
entially into one of the phases of the lipid mixtures. Also, Laurdan
fluorescence behavior is less sensitive toward GM1-containing compo­
sitions due to the complexity of the polar head group [51].
R. Tamulytė et al.
Fig. 2. The normalized Laurdan fluorescence emissions of LUVs recorded at 22, 37, and 60 ◦ C temperature: (a) brain total lipid extract; (b) DOPC/DOPE/bSM/Chol/
GM1 (44/20/30/5/1 mol%); and (c) the generalized polarization (GP). The Laurdan probe to lipid ratio was 1/200.
3.3. S100A9 self-assembly in the presence of liposomes
The ability of S100A9 to self-assemble into fibrils may lead to the loss
of signaling functions and acquired amyloid cytotoxicity [2]. The
Thioflavin-T (ThT) assay is a standard method to quantify the growth of
fibrillary aggregates during amyloid formation [36,52]. ThT, as an
extrinsic fluorescent probe molecule, once bound to the protein’s
β-structure, gives a characteristic fluorescent emission at ~480 nm (λexc
440 nm). The ThT intensity is related to the growth and amount of
fibrillar structures. Fig. 3 displays the increase in fluorescence intensity
of S100A9 bound-ThT over time in the presence or absence of liposomes.
We incubated 100 μM of S100A9 protein w/o of 500 μM unilamellar
liposomes at 37 ◦ C pH 7.2 under orbital agitation to evaluate if the
presence of the lipid surface promotes the protein accumulation. The
high protein concentrations were needed to accelerate the slow aggre­
gation process. Still, the protein concentration was probably insufficient
as the plateau was not reached within our experiment’s timeframe (75
h).
The changes in ThT fluorescence intensity indicate a slight depen­
dence of the number of formed fibrillary structures on the lipid
composition. The sequence from the slowest to the fastest aggregation is
DOPC/DOPS < DOPC/DOPE/Chol/bSM/GM1; DOPC/DPPC; DOPC/
Chol < S100A9 alone < BTLE. At the initial stages (first 24 h) of accu­
mulation, all lipid compositions had lower ThT intensity than protein
alone. However, after 35 h of incubation, the intensity data for BTLE
liposomes increased significantly and stood out from the rest composi­
tions. While BTLE liposomes promoted protein aggregation, the aggre­
gation of S100A9 in the presence of negatively charged liposomes was
reduced. Meanwhile, zwitterionic, two-phase, and five-component lipid
compositions slightly inhibit fibrillation compared to the S100A9 pro­
tein alone.
5
BBA - Biomembranes 1865 (2023) 184113
We would expect that the size of the liposome should be affected by
the protein’s binding. The DLS data (see Supplementary materials
Fig. S8 and Table S2) indicate that liposomes in a protein-free envi­
ronment remain stable over 72 h. An exception is the DOPC/DPPC
composition, for which a steady state was reached after the increase of
the diameter of liposomes by 36 nm in the first 24 h. However, we
observe a 20-fold increase in the protein’s hydrodynamic diameter,
while changes in BTLE and DOPC/Chol liposome size are insignificant
(see Table 1). The surface presence should increase local concentration,
promote binding of the material, and further accumulation. Important to
mention that factors such as curvature, lipid phase, and electrostatic
interaction are also involved in this process. However, a lower number
of S100A9 aggregates in the presence of liposomes reveals that the
nucleation process is disturbed. Likely, the binding to the lipid surface is
limited due to the hydrophilic surface of S100A9 in a calcium-free
environment.
The observed (Fig. 3) short and not-well-defined lag phase represents
the time required for the nuclei formation. Our results agree with pub­
lished reports that in vitro S100A9 protein alone self-assembles into
amyloids by the nucleation-dependent polymerization mechanism
[53,54]. This unusual behavior for other amyloid-like proteins indicates
that nucleation sites are small and fast-forming. In comparison to the
S100A9 published data [53,54], our measurements were performed
under similar physiological conditions: PBS buffer (pH 7.2 vs. 7.4),
temperature (37 vs. 42 ◦ C), and calcium-free environment. The main
difference is that we promoted aggregation by external agitation. It
should be noted that calcium ions significantly slow down the S100A9
oligomerization [55] (see Supplementary materials Fig. S9).
Fig. 3. Monitored aggregation of S100A9 (100 μM)
by Thioflavin-T (ThT) fluorescence: (a) for S100A9
protein alone (black) and in the presence of lipid
vesicles composed of DOPC/Chol (blue), DOPC/
DOPS (cyan), DOPC/DPPC (purple), DOPC/DOPE/
bSM/Chol/GM1 (red) and BTLE (green); monitoring
time 75 h; (b) enlarged inset from the (a) panel;
monitoring time 24 h. Experimental details: PBS
buffer (pH 7.2), at 37 ◦ C, w/o 500 μM of lipid. Con­
trol: protein samples without lipids but under other­
wise identical conditions. Each dot represents the
average ± S.D. from triplicate measurements.
R. Tamulytė et al. BBA - Biomembranes 1865 (2023) 184113

3.4. Visualization of S100A9-induced lipid membrane remodeling by Table 2

AFM The changes of the arithmetic average roughness (Image Ra) of repre­
sentative AFM images of tBLMs incubated with S100A9.
Our ThT fluorescence data indicates that the zwitterionic DOPC/ Sample Ra, nm
cholesterol 60/40 mol% lipid bilayer does not affect or slightly inhibit DOPC/Chol (Fig. 4) 0.35
the aggregation. Due to high cholesterol content and membrane local + S100A9 (0.5 h) –
stiffening [56], tethered bilayer lipid membranes (tBLMs) would form a + S100A9 (2 h) 0.35
lamellar liquid-ordered (Lo) phase [46,47]. The influence of membrane DOPC/DOPS (Fig. 4) 0.38
+ S100A9 (0.5 h)
fluidity on lipid-protein interaction is the object of interest as mem­ –
+ S100A9 (2 h) 0.40
branes’ fluidity decreases with cells’ age [57]. Fig. 4a–c panels show a DOPC/DPPC (Fig. 6) 0.46
series of AFM topographic images of prepared tBLMs recorded before + S100A9 (0.5 h) 0.47
and after 2 and 24 h incubation with S100A9 protein. No significant + S100A9 (2 h) 0.54
morphology changes in the lipid layer were observed even after incu­ DOPC/DOPE/Chol/bSM/GM1 (Fig. 7) 0.91
+ S100A9 (0.5 h) 0.75
bation for 24 h (Fig. 4c). Probably due to too low protein concentration, + S100A9 (1 h) 0.68
slower aggregation at room temperature, or localized accumulation
overlooked by AFM imaging. The roughness value (Image Ra) for the
area imaged in Fig. 4a is 0.35 nm and remains constant over 2 h negatively charged surface. However, the driving force of this effect is a
(Table 2). lower surface pH compared to the bulk solution. The attraction of
Next, we prepared lipid bilayers with negatively charged phospha­ counterions as H+ could lower the local pH by up to 2-fold [59,60]. The
tidylserine (PS). PS lipid is actively confined to reside solely in the inner local decrease in pH was assumed to denature the native conformation
cytoplasmic leaflet of the plasma membrane lipid bilayer. It is only of proteins (reducing solubility), inducing the formation of acidic
transferred to the outer lipid layer in conditions like apoptosis and molten globule states and their interaction with or insertion into
cancer [58]. Fig. 4d–f panels show AFM topographic images of tBLMs membranes [60,61]. These results contradict ThT fluorescence data, as
formed from MLVs of DOPC/DOPS (60/40 mol%). In contrast to DOPC/DOPS liposomes had the lowest intensity values and the number
cholesterol, PS accelerates the accumulation of S100A9 protein on of aggregates suggesting that protein might be spontaneously incorpo­
membrane surfaces, as seen in the AFM images (Fig. 4f and g panels). rated into the bilayer [62]. Therefore, protein is no more accessible for
After 24 h of incubation in the presence of S100A9, the visible white nucleation. In this study, it was impossible to distinguish whether it was
blobs indicate globular aggregates of 4–10 nm height (Fig. 4h). The a partial or complete insertion. At room temperature, DOPC/DOPS li­
calculated pI value for S100A9 protein is 5.7 and at pH 7.2 has a posomes are in a liquid-disordered phase [48]; in comparison, DOPC/
negative net charge of − 6.11, suggesting no possible interactions with a Chol liposomes are in a liquid-ordered phase. Nevertheless, the

Fig. 4. AFM topography of pristine tBLMs and incubated with 5 μM of S100A9. Lipid bilayers were formed from DOPC/Chol 60/40 mol% (a) and DOPC/DOPS 60/
40 mol% (d) multilamellar vesicles. AFM images show changes in topography following incubation for 2 h (b and e) and 24 h (c and f), respectively. The scale bar is
400 nm. In a magnified view (g) from (f), the scale bar is 140 nm. The (h) panel represents cross-section profiles (1 and 2).

6
R. Tamulytė et al.
morphology observable in the AFM images showed that these two-
component liposomes form homogeneous tethered lipid bilayers. Thus,
the differences observed between these two compositions might also be
related to the fluidity of the lipid bilayer and reduced barrier between
the acyl chains, which presumably affects initial protein binding.
Cell membranes have highly heterogeneous lipid compositions. To
better simulate the healthy neuronal membrane, we used a membrane
comprised of natural brain total lipid extracts (BTLE), consisting of over
58 % unidentified lipids. The registered GP value at room temperature
for BTLE is slightly higher than for DOPC/cholesterol (see Supplemen­
tary materials Fig. S7); we assume BTLE liposomes have a similar lipid
packing. Therefore, Laurdan fluorescence and AFM data (Fig. 5a) indi­
cate that the lipid bilayer formed from the extract is in a monophasic
liquid-ordered phase. Also, 15 wt% of the extract’s lipids have anionic
headgroups. Likely, as in the DOPC/DOPS case, the protein accumula­
tion will be accelerated based on reduced local pH. The highest regis­
tered ThT fluorescence confirmed that. However, the topography of
lipid BTLE bilayers showed apparent changes after adding S100A9
protein (Fig. 5). Following the protein incubation, spontaneous defects
were formed. The depressed regions’ average depth is 1.31 ± 0.23 nm
(n = 12) (Fig. 5g). Following the longer incubation times, as seen in
Fig. 5 e and f panels, the expansion of these reduced step heights is
observed. The depth increases up to 2.20 ± 0.29 nm (n = 12) (Fig. 5h),
and for a better overview, the large-scale AFM images are presented in
Fig. S4 (see Supplementary materials). The thickness of the initial
bilayer is approx. 5.2–5.3 nm (Fig. S3), in agreement with previous re­
ports for BTLE-supported lipid bilayers (SLBs) [63]. The depth of formed
pits is less than one leaflet of a lipid bilayer. Thus some structural
changes and the formation of compressed layers occurs. A similar
decrease in depth by ~1–2 nm was reported for α-synuclein [64,65] and
antimicrobial peptide melittin [66], as well as alcohols [67] incubated
with lipid bilayers. The cause is either the removal of the upper leaflet by
a detergent-like effect or thinning of acyl lipid chains and the formation
of interdigitated domains caused by peptide binding. Removal of a distal
leaflet and exposure of hydrophobic tails into the water environment is
energetically unfavorable and is stabilized by protein coverage of
exposed lipid acyl chains. However, the formation of detectable protein
blobs within compressed valleys was not observed. More likely, S100A9
induces local membrane thinning but not the removal of lipids from the
Fig. 5. Topographic investigation of BTLE lipid bilayer before (a) and after injection of 5 μM of S100A9. The protein causes the formation of defects (indicated by
arrows in the b panel). Different locations of the same sample were visualized after 0.5 h (b, c), 1 h (d), and 2 h (e and its magnified view in the f panel, the scale bar is
140 nm) of incubation. Profiles in the g and h panels are from the b and f panels, respectively. The image size is 2 μm. The Z-range is 10 nm.
7
BBA - Biomembranes 1865 (2023) 184113
distal leaflet. Since we observed that the initial depth increases from
1.31 ± 0.23 nm to 2.2 ± 0.29 nm, it shows the progression of membrane
compression [67] by increasing the protein’s local concentration over
time. Here an evident lateral expansion of lipids, with further insertion
into the membrane, is associated with a decrease in the thickness of the
bilayers. The significant increase in protein aggregates in the presence of
brain total lipid extract liposomes compared to pure S100A9 solution
(Fig. 3) is somehow related to these drastic changes in the bilayer
integrity.
In eukaryotic cells, fluidity across the membrane is not homogenous.
Lipid mixtures with different melting temperatures induce the formation
of localized lipid packing [68] and mimic liquid-ordered (Lo) and
disordered (Ld) regions on a solid support lipid bilayer. Domains of
different fluidity show reduced or increased thickness. The cantilever
tip-induced compression of the lipid bilayer in AFM topographies ob­
tained in the conventional tapping mode enhances the height difference
[69]. Fig. 6 shows topography and phase images of S100A9 protein
incubated with tBLMs, formed from DOPC and a saturated lipid DPPC
(60/40 mol%) MLVs. As shown in Fig. 6a, the topographic image ob­
tained for the protein-free bilayer revealed the coexistence of two phases
at room temperature. The DOPC acyl chains are fluidic at room tem­
perature and are in the liquid-crystalline phase (Lα or Ld). The melting
temperature of acyl chains for saturated lipid DPPC is 41 ◦ C. Thus, the
higher areas are assigned to DPPC-enriched domains at room tempera­
ture and are in the gel state (Lβ or So) [49]. The differences between
these two phases are also visible in the phase images (Fig. 6, e–h panels),
where the stiffer domains are brighter than the surrounding. The
observed step height difference between phases of different mechanical
properties is 1.04 ± 0.30 nm (n = 11), comparable to the confirmed
height difference from AFM data for SLBs (0.8–1.1 nm) [70,71]. Before
the protein’s addition, the area of interest was scanned for at least an
hour to confirm the layer’s morphological stability (see Fig. S10).
S100A9 protein induced a slow disappearance of the gel phase domains
(brighter regions in AFM image), which was completed after 2 h. The
erosion of Lβ domains started from the edges as it was more exposed to
the environment.
Interestingly, no further morphological alterations (i.e., holes) were
observed apart from the slow disappearance of these protrusions.
Similar lipid bilayer remodeling was observed for the dicationic
R. Tamulytė et al. BBA - Biomembranes 1865 (2023) 184113

Fig. 6. AFM topography (upper panels) and phase (lower panels) images of phase-separated lipid bilayers. Time-lapse images represent bilayer changes after
exposure to S100A9 for 2 h: (a and e) intact tBLM was formed from MLVs DOPC/DPPC 60/40 mol%; (b and f) after 0.5 h; (c and g) after 1 h, (d and h) after 2 h. The
scan size is 3 μm × 3 μm. In AFM topography images, the color scale indicates the height of the observed structures. In the chosen color scheme, the brighter areas
(emerald green) show the protrusion of higher objects. The DOPC-enriched regions are marked as Ld, and the DPPC-rich domains are Lβ.

antibiotic azithromycin [72] or surfactin [71]. This effect was attributed
to the disruption of the tight molecular packing of the DPPC molecules.
The erosion of the gel phase is reflected in the surface roughness
decrease (Table 2). However, compared to monophasic DOPC/Chol or
DOPC/DOPS tBLMs, the Image Ra value for Fig. 6d is higher, 0.35–0.38
and 0.46, respectively. In the enlarged views (Supplementary materials
Fig. S11), we observe that after protein addition, not only do the DPPC
domains tend to dissolve, but small fragments populate the lower lipid
bilayer. However, the observed morphological changes were not
assigned as lipid surface-bound proteins. Due to the rough tBLMs sur­
face, only larger structures could be appropriately distinguished.
The formation of solid domains in living systems is vital for cellular
processes; these structures are also called rafts. It was reported that
matured S100A9 oligomers bind with one of the lipid-raft components,
ganglioside (GM1) [27]. Thus, we chose to imitate the outer layer of the
neuronal plasma membrane [30] by forming tBLMs from DOPC/DOPE/
bSM/Chol/GM1 44/20/30/5/1 mol% multilamellar vesicles. This
composition at room temperature should spontaneously lead to liquid-
ordered–disordered phase separation, with ordered domains enriched in
brain SM, cholesterol, and GM1 [73]. Fig. 7a represents the AFM
topography image of two coexisting immiscible areas. The darker
brownish areas are assigned to fluid phosphatidylcholine (Ld), and the
brighter ones are bSM-cholesterol-GM1-enriched solid domains (Lo).
The formed domains are higher than the surrounding area by 2.5 ± 0.2
nm (n = 5). Time-scale AFM images presented in Fig. 7 show the shape
and evolution of these domains over time. It is important to note that the
GM1 residues have a negative charge, and the height of the domains
could be influenced by the applied force [69] and the electrostatic

Fig. 7. AFM topography and profiles of lipid raft-like domains in tBLMs formed on mica substrate. tBLMs were formed from DOPC/DOPE/bSM/Chol/GM1 44/20/
30/5/1 mol% multilamellar vesicles. AFM image (a) of an intact lipid bilayer and profile of the lipid raft domain (e). Images and profiles (b and f), (c and g), and (d
and h) show topography changes following 0.5 h, 1 h, and 24 h of incubation with S100A9 protein, respectively. Images visualized in PeakForce QNM mode. The
scale bar is 200 nm. The DOPC-enriched regions are marked as Ld, and SM/cholesterol/GM1-rich regions — as Lo.

8
R. Tamulytė et al.
interaction of the AFM tip [74]. Also, in this work, two types of AFM
modes were used: tapping and PeakForce QNM. Due to precise force
control, the rigid domains would appear with higher height differences
in images obtained by PeakForce QNM than in tapping mode. The
similar height difference between Ld and GM1-containing Lo phases was
observed and described before (2.2 ± 0.2 nm [75]; 2.4 ± 0.3 nm [76]).
The GM1 headgroup has five residue branch oligosaccharides. Given the
size of a glucose molecule, approx. 0.8 nm in the long axis (the average
carbon‑carbon bond length is 0.13 nm [77]), and the length of the GM1
oligosaccharide headgroup can be estimated at roughly 2.5–3.0 nm. The
height difference between DPPC-containing or GM1-containing domains
and the lower surrounding areas for tBLMs is comparable to data for
SLBs formed on smooth mica substrate [70,71,75,76]. Also, the di­
mensions of observed raft-like domains are smaller than those of Lβ
domains in DOPC/DPPC tBLMs. Likely, the appearance of domains in
tBLMs, including size distribution and shape, is highly dependable on
the substrate (i.e., the impact of gold-coated mica). This issue was also
described in detail [78]; the domains formed on glass substrate
compared to structures obtained on smooth mica are smaller with
irregular boundaries. The molecular diffusion remains the same on the
different substrates, and the authors proposed a significant hindrance of
hydrodynamic lipid flow resulting in restricted domain growth [78].
Fig. 7b and c panels show the same area during the first hour of in­
cubation of raft-like tBLMs with S100A9 protein. After adding S100A9
protein, we observed a decrease in the domains’ height by 1 nm from 2.5
± 0.2 nm to 1.5 ± 0.2 nm (n = 6). The average step height of the same
sample after incubation with protein for 24 h was 1.5 ± 0.4 nm (n = 6)
(Fig. 7, d and h panels). Fig. S5 (see Supplementary materials) shows
differences between the large-scale time-lapse AFM images. We observe
not only the reduction in height but surface roughness decreases from
0.91 nm to 0.68 nm for pristine tBLMs and incubated with protein for 1
h, respectively. We hypothesize that these alterations indicate the sol­
ubilization of raft domains. Also, we observed that the domains’ sur­
rounding areas changed their appearance. Previously smooth Ld regions
became rougher due to bound protein or the formation of protein-
induced lipid-raft nanosized domains. A lipid rafts’ fragmentation was
already demonstrated for human α-synuclein [79] incubated with a
reconstituted membrane bilayer and was observed for the myristoyl
modification of tyrosine kinase c-Src [80]. Hence, based on these results,
we speculate that the protein can recruit lipid molecules to avoid un­
favorable hydrophobic mismatch.
Finally, in our experiments, we also applied a calcein leakage assay.
Unfortunately, control liposomes were unstable under experimental
conditions (see Supplementary materials, Fig. S2), with at least 20 %
calcein content loss over 4 h of measurement. A similar permeability is
observed for liposomes incubated with protein. Thus, the protein did not
promote any membrane leakage. The hydrodynamic radius of calcein is
0.7 nm, and only defects exceeding 1.4 nm in diameter are detectable in
calcein release measurements.
4. Concluding remarks
Although the role of the S100A9 protein in Alzheimer’s disease re­
mains debated, the interaction of the protein with cellular membranes
seems essential for both functionality and toxicity. In this study, we
successfully applied AFM to investigate the impact of the lipid packing,
the headgroup charge, and the incorporation of GM1 in tethered lipid
bilayers on the binding and accumulation of S100A9 in a calcium-free
environment. However, the approach used in our work has several
limitations. AFM data is not sufficient to determine whether the
morphological alterations caused by protein actions affect the lipid bi­
layer’s permeability. Therefore, more sensitive techniques like electro­
chemical impedance spectroscopy should be applied [81]. Similar
topographical effects may indicate different mechanisms of protein ac­
tion. Despite these limitations, we have determined the morphological
changes associated with exposing lipid bilayers to S100A9. Based on our
9
BBA - Biomembranes 1865 (2023) 184113
observations, the phase state or charge of the membrane affects protein-
lipid interaction. These results support three models for membrane-
mediated S100A9 toxicity: accumulation on a negatively charged sur­
face, gel or liquid-ordered phase disruption, and lipid layer thinning.
The most drastic changes in the loss of lipid bilayer’s integrity were
registered for brain total lipid extract lipid bilayers.
In conclusion, despite the visible changes in the bilayer’s
morphology, the presence of membrane did not lead to the severe pro­
motion of protein aggregation. The binding of Ca2+ ions changes the
protein’s conformation; thus, future works should focus on more bio­
logically relevant conditions to expand the knowledge of the S100A9
protein’s mechanistic role in the progression of diseases. Furthermore,
the structural data that protein undergoes upon aggregation impacts the
interaction with lipid bilayer mechanism are also lacking.
CRediT authorship contribution statement
R.T. carried out the experiments, analyzed the data, and wrote the
original draft.
M.J. designed the experiment and wrote the paper.
E.J. purified the protein and reviewed the paper.
Z.T. purified the protein and reviewed the paper.
V.S. formal analysis, resources.
All authors read and approved the final manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Authors gratefully acknowledge Dr. Darius Šulskis (Institute of
Biotechnology, Life Sciences Center, Vilnius University) for his help in
the S100A9 protein purification, and Prof. Gintaras Valinčius (Institute
of Biochemistry, Life Sciences Center, Vilnius University) for support.
We acknowledge the author of tBLMs’ schematic view (Fig. S6) — Dr.
Tadas Penkauskas (Institute of Biochemistry, Life Sciences Center, Vil­
nius University).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbamem.2022.184113.
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