Cellular and Molecular Life Sciences(2023) 80:167
https://doi.org/10.1007/s00018-023-04813-9

Cellular and Molecular Life Sciences

ORIGINAL ARTICLE

Cell density‑dependent membrane distribution of ganglioside GM3
in melanoma cells
Motohide Murate1,2,3 · Noriko Yokoyama4 · Nario Tomishige1,2,3 · Ludovic Richert2 · Nicolas Humbert2 ·Brigitte Pollet2 · Asami Makino1,5 · Nozomu Kono6 · Laura Mauri7 · Junken Aoki6 · Yasushi Sako3 · Sandro Sonnino7 ·Naoko Komura8 · Hiromune Ando8 · Mika K. Kaneko9 · Yukinari Kato9 · Kei‑ichiro Inamori10 · Jin‑ichi Inokuchi10,11 ·Yves Mély2 · Kazuhisa Iwabuchi4 · Toshihide Kobayashi1,2,3
Received: 20 January 2023 / Revised: 21 April 2023 / Accepted: 17 May 2023
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023

Abstract
Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of
GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was
shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane
environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover,fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low
density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing
phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody
senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.
Keywords Anti-ganglioside antibodies · Crypticity · Fluorescence lifetime imaging · Lipidomics · Plasma membrane

Toshihide Kobayashi: Lead contact.

Abbreviations
Chol	Cholesterol
DMPC	1,2-Dimyristoyl-sn-glycero-3-phosphocholine
DOPC	1,2-Dioleoyl-sn-glycero-3-phosphocholine
6* Kazuhisa Iwabuchi
iwabuchi@juntendo.ac.jp

Department of Health Chemistry, Graduate Schoolof Pharmaceutical Sciences, The University of Tokyo, 7‑3‑1,
Hongo, Bunkyo‑Ku, Tokyo 113‑0033, Japan7* Toshihide Kobayashi
toshihide.kobayashi@unistra.fr

Department of Medical Biotechnology and Translational
Medicine, University of Milan, Milan, Italy

8

Institute for Glyco‑Core Research, Gifu University, 1‑1
Yanagido, Gifu 501‑1193, Japan* Motohide Murate
mmurate@riken.jp

1

Lipid Biology Laboratory, RIKEN, Wako,
Saitama 351‑0198, Japan

9

2

Laboratoire de Bioimagerie et Pathologies, UMR 7021,
CNRS, Faculté de Pharmacie, Université de Strasbourg,
67401 Illkirch, France

Department of Antibody Drug Development, Tohoku
University Graduate School of Medicine, Sendai,
Miyagi 980‑8575, Japan

10

Division of Glycopathology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical
and Pharmaceutical University, Sendai 981‑8558, Japan

11

Forefront Research Center, Graduate School of Science,
Osaka University, Toyonaka, Osaka 560‑0043, Japan

3

Cellular Informatics Laboratory, RIKEN CPR, Wako,
Saitama 351‑0198, Japan

4

Institute for Environmental and Gender‑Specific Medicine,
Graduate School of Medicine, Juntendo University, Urayasu,
Chiba 279‑0021, Japan

5

Molecular Physiology Laboratory, RIKEN CPR, Wako,
Saitama 351‑0198, Japan

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DPPC	1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
EGFR	Epidermal growth factor receptor
F2N12S	N-[[4’-N,N-diethylamino-3-hydroxy6-flavonyl]-methyl]-N-methyl-N(3-sulfopropyl)-1-dodecanaminium: inner salt
FLIM	Fluorescence lifetime imaging microscopy
FRET	Förster resonance energy transfer
GlcCer	Glucosylceramide
GM1	Ganglioside GM1
GM3	Monosialoganglioside GM3(NeuAcα3Galβ-4Glcβ1Cer)
Ld	Liquid-disorderedLo	Liquid-ordered
MDCK	Madin-Darby canine kidney
NPA	Niemann-pick A
POPC	1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
pSM	Palmitoyl sphingomyelin
SM	Sphingomyelin
SPR	Surface plasmon resonanceIntroduction
Monosialoganglioside GM3 (NeuAcα3Galβ-4Glcβ1Cer)is the simplest ganglioside. GM3 is distributed in various
mammalian cells and tissues and is involved in multiple cellular signaling by modulating the activities of integrin [1],insulin receptor [2] and epidermal growth factor receptor(EGFR) [3, 4]. It is postulated that cell surface distributionof GM3 is crucial for its function [5]. Monoclonal anti-GM3
antibodies [6–8] and polyclonal anti-GM3 antibodies frompatients [9] have been used to examine cellular GM3 distribution. These antibodies revealed heterogeneous distributionof GM3-rich domains on the plasma membrane of human
T cells [9], Madin-Darby canine kidney (MDCK) cells [10]and mouse fibroblasts [11]. It was also demonstrated that
GM3-rich domains were distinct from caveolae in MDCK
cells [12].
Anti-ganglioside antibodies are believed to be a crucial
pathogenic factor of peripheral nerve diseases [13]. It is wellrecognized that antigen gangliosides are often cryptic [14].
“Cryptic glycolipids” are the cell surface glycolipids that are
not accessible to proteins such as anti-glycolipid antibodies or sialidases. This makes the evaluation of pathogenic
antibodies and determination of ganglioside distributiondifficult. Currently, two mechanisms are reported for theganglioside crypticity: (1) steric hindrance by other gangliosides for the binding of anti-GM3 [15] and anti-GM1 [16]antibodies and (2) cholesterol-induced conformation change
of GM1 [17]. It is not known whether other mechanism(s)might also be involved in ganglioside crypticity.

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Previously, it was shown that anti-GM3 antibody labeledsparse but not high density B16 melanoma cells [18]. Similar results were obtained in hamster embryo fibroblasts [18]and glioma cells [19]. These cells contain GM3 as a single
ganglioside. Cell density did not affect cellular GM3 content[18] but may affect its distribution in sparse and confluent
melanoma and glioma cells. Surface labeling of GM3 inglioma cells by sodium ­[3H] borohydride after sodium peroxide treatment indicated that cell surface GM3 was equallylabeled in sparse and confluent cells [19]. These results indicate that GM3 is cryptic in confluent melanoma and glioma
cells and suggest different membrane organization of GM3
in sparse and confluent melanoma cells.
In this study, we investigated the interaction of monoclonal anti-GM3 antibodies with GM3 in different membrane
environments using model membranes and cultured cells.
Our results indicate that the antibodies preferentially bind
to GM3 clusters. Formation of GM3 cluster is dependent on
membrane fluidity and the plasma membrane is more fluid in
confluent melanoma cells as compared to sparse cells. Thus,
our studies revealed a cell density-dependent alteration of
membrane organization of GM3.

Results
Binding of anti‑GM3 antibody to GM3/partner
lipid is dependent on incubation temperaturesand phase transition temperatures of partner lipids
Mouse monoclonal IgM against GM3 (clone GMR6) established by Kotani et al. [7] by immunizing purified GM3,
has been widely employed to label cellular GM3 [10–12,
20–25]. We first characterized this antibody in depth using
model systems. In Fig. 1A, the binding of the antibody tovarious lipids was compared. The antibody strongly boundto milk GM3 (GM3) and to a lesser extent to GM2. The
antibody did not significantly bind to other glycolipids or
phospholipids, including lyso GM3.
In Fig. 1B, GM3 was mixed with 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC, Gel to liquid crystalline phasetransition temperature, T­ m =  − 17 ℃), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, T­ m =  − 2 ℃),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC,­Tm = 24 ℃) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, T­ m = 41 ℃) [26] and the binding of the antiGM3 antibody was measured at 4 ℃, room temperature(23 ℃), 37 ℃ or 42 ℃ by ELISA. The antibody efficiently
bound GM3/DPPC (1:9) at 4 ℃ and room temperature.
The binding was drastically decreased at 37 ℃ and 42 ℃.
Although the binding was less efficient, binding to GM3/
DMPC (1:9) and GM3/POPC (1:9) also showed temperature dependence. The antibody did not significantly boundCell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	

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B

OD490 – OD630

OD490– OD630

A

Page 3 of 15CF
GM3/DPPC

22.2 nM
11.1
5.6

GM3/DOPC

2.8
0.56
0.06

E
+Chol

1.1

OD490 – OD630

DTime (sec)-CholFig. 1  Binding of anti-GM3 antibody (GMR6) to GM3/partner
lipid is dependent on incubation temperatures and phase transition
temperatures of partner lipids. A Binding of 0.37 µg/mL of antiGM3 antibody to indicated lipids (0.5 nmol/well) was measured by
ELISA. GM1, ganglioside GM1; GM2, ganglioside GM2; GM3,
ganglioside GM3; GlcCer, glucosylceramide; LacCer, lactosylceramide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; pSM, palmitoyl sphingomyelin;Cer,ceramide; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); TOCL, 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol; CerPE, N-acyl-sphingosylphosphorylethanolamine. B Binding of 0.37 µg/mL of anti-GM3
antibody to GM3/DOPC (1:9), GM3/POPC (1:9), GM3/DMPC (1:9)
or GM3/DPPC (1:9) (0.5 nmol GM3/well) at indicated temperature
measured by ELISA. (C) Binding of 0.37 µg/mL of anti-GM3 antibody to GM3/pSM (1:9), GM3/GlcCer (1:9), GM3/GM1 (1:9) or
GM3 alone (0.5 nmol GM3/well) at indicated temperatures measured by ELISA. Data in A–C are means ± SD of three experiments.
D Binding of anti-GM3 antibody (10 ng/mL) to GM3/DOPC (1:5)(orange) or GM3/DPPC (1:5) (green) liposomes measured by surface
plasmon resonance (SPR). SPR was performed as described in Methods. E Binding of anti-GM3 antibody to DPPC/GM3 (5:2) (orange)or DPPC/GM3/Chol (5:2:5) (green) liposomes. F Binding of different
concentration of anti-GM3 antibody to GM3/DPPC (1:9) liposomes

to GM3/DOPC (1:9). In Fig. 1C, we examined the effect ofpalmitoyl sphingomyelin (pSM, T­ m = 40 °C), glucosylceramide (GlcCer, ­Tm = 66–68 ℃) and ganglioside GM1 (GM1,­Tm =  ~ 20 ℃) [26] on the antibody binding when mixed with
GM3. Binding to GM3/pSM (1:9) and GM3/GM1 (1:9)
showed similar results to those of GM3/DPPC and GM3/
DMPC. In contrast, the binding to GM3/GlcCer (1:9) did
not show clear temperature dependence between 4 and 42 ℃.
Binding to pure GM3 (­ Tm = 35–40 ℃) [26] also did not showtemperature dependence (Fig. 1C). These results indicatethat the binding of anti-GM3 antibody to GM3-containing
lipid mixture is dependent on the physical properties of thepartner lipids and that gel phase lipids give higher binding
than liquid crystalline lipids. In Suppl. Fig. 1, we measured
binding of anti-GM3 antibody to synthetic GM3s (sphingosine backbone C18:1, fatty acid composition C8:0 to C24:0).
The antibody bound efficiently to C16:0 GM3 and C24:0
GM3. Binding was decreased in C12:0 GM3. The antibody
did not bind to C8:0 GM3. Similar to milk GM3, in C12:0
GM3 to C24:0 GM3, the antibody bound to GM3/DPPC(1:9) more efficiently than GM3/POPC (1:9).
We then compared the binding of other established antiGM3 monoclonal antibodies to GM3/DPPC and GM3/
DOPC. Anti-GM3 IgM monoclonal antibody M2590 was

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established after immunization of C57BL/6 mice with syngenic B16 melanoma cells [6, 27]. Anti-GM3 IgG monoclonal antibody DH2 was originally established after immunization of C57BL/6 mice with GM3 lactone coated on
Salmonella minnesotae [8]. The DH2 antibody is a mouse­IgG3 known as a highly sensitive and specific antibody
against GM3. However, in recent years, antibody production
from DH2 hybridoma has become unstable, and the activityof purified antibody has been low. Therefore, we cloned theantibody gene from the DH2 hybridoma and constructed
expression plasmids for the H and L chains. The recombinant DH2 (recDH2) antibody purified from supernatantculture of CHO/recDH2 cells was used for binding study.
Both antibodies showed higher binding to GM3/DPPC
and lower binding to GM3/DOPC (Suppl. Fig. 2). These
results indicate that the partner lipid-dependent binding to
GM3 is a common property of anti-GM3 antibodies. We
used GMR6 antibody in the following experiments.
Next, we examined the binding of the antibody to immobilized lipid bilayers containing GM3 by surface plasmon
resonance (SPR) (Fig. 1D–F). As observed in ELISA,
the binding of GMR6 antibody was significantly different
between GM3/DPPC and GM3/DOPC (Fig. 1D). Previously,
the binding of cholera toxin B-subunit to GM1 was shown
to be inhibited by cholesterol (Chol) [17]. Thus we added
Chol to GM3/DPPC liposomes. The addition of Chol did
not affect the binding of the antibody to GM3 (Fig. 1E).
Consistent with SPR results, presence of Chol did not inhibit
the antibody binding to GM3 in ELISA (Suppl. Figure 3).
These results suggest that the molecular mechanism of
GM3/DOPC crypticity against anti-GM3 antibody is different from that of GM1/Chol to cholera toxin. From Fig. 1F
the dissociation constant K­ D of GMR6 antibody to GM3/
DPPC (1:9) was calculated to be 2.8 ± 0.8 × ­10–10 M (n = 4).
The binding of the antibody to GM3/DOPC was too low to
obtain reliable ­KD value.

Fluorescent GM3 analogs form clusters in DPPC
To examine the difference of GM3 distribution in DPPC andDOPC membranes, we employed two fluorescent GM3 analogs, C11 TopFluor-GM3 and ATTO594-GM3 [28] (Suppl
Fig. 4). Fluorescent lipid analogs exhibit self-quenching ina concentration-dependent manner [29, 30]. There is little self-quenching if small amount of a fluorescent lipid iswell mixed with bulk membrane lipids. In contrast, when afluorescent lipid is excluded from bulk lipids, fluorescentlipid forms clusters and thus is self-quenched [31, 32]. Solubilization of lipids by detergent dilutes fluorescent lipid
clusters in detergent micelles and thus dequenches fluorescence. We first measured emission spectra of DOPC/C11
TopFluor-GM3 (99.5/0.5 mol/mol) (Fig. 2A) and DPPC/

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M. Murate et al.

C11 TopFluor-GM3 (99.5/0.5 mol/mol) (Fig. 2B) in theabsence and presence of 1% Triton X-100 at 20 ℃. C11
TopFluor-GM3 was quenched 21% in DOPC and 44% in
DPPC. Comparison of Fig. 2A and B shows that even in
the presence of Triton X-100, the fluorescence intensity of
DOPC/C11 TopFluor-GM3 is higher than that of DPPC/C11
TopFluor-GM3. This may be due to the partial insolubilityof DPPC in Triton X-100 [33]. Thus we may underestimate
the quenching efficiency of C11 TopFluor-GM3 in DPPC.
These results suggest that in both DOPC and DPPC, TopFluor is clustered. However, the number and/or size of clusters are larger in DPPC.
There is an overlap in the emission spectrum of C11 TopFluor-GM3 and the absorption spectrum of ATTO594-GM3.
Thus we can expect Förster resonance energy transfer(FRET) between the two lipids when they are in close proximity. Figure 2C and D show emission spectra of DOPC andDPPC liposomes containing 0.5 mol% C11 TopFluor-GM3
and 0.5 mol% ATTO594-GM3, respectively, in the absence
and presence of 1% Triton X-100 at 20 ℃. Appearance of
630 nm peak in the absence of Triton X-100 indicates FRET
between C11 TopFluor-GM3 and ATTO594-GM3. Calculation of fluorescence intensity at 507 nm in the presence andabsence of FRET acceptor, ATTO594-GM3, in the absenceof Triton X-100 using Eq. (1) indicates that FRET efficiency
is 64% in DOPC and 82% in DPPC. We then measured selfquenching of ATTO594-GM3 in these liposomes by measuring fluorescence from 605 to 700 nm at excitation 603 nm in
the presence and absence of Triton X-100 (Fig. 2E and F).
ATTO594-GM3 was self-quenched 10% in DOPC and 50%
in DPPC. Similar to Fig. 2A and B, fluorescence intensityof DPPC/ATTO594-GM3 showed lower fluorescence than
DOPC/ATTO594-GM3 in the presence of Triton X-100.
Increased self-quenching of ATTO594-GM3 and increased
FRET in DPPC suggest that DPPC enhanced clustering of
GM3 analogs.
We then measured emission spectra of DOPC/C11
TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5 mol/mol/mol)(Fig. 2G) and DPPC/C11 TopFluor-GM3/ATTO594-GM3(99/0.5/0.5 mol/mol/mol) (Fig. 2H) in the absence of 1% Triton X-100 at different temperatures. In DPPC, FRET signal
was higher at 4 ℃ and 20 ℃ compared to 37 ℃ and 42 ℃
(Fig. 2H–J). In contrast, DOPC did not show temperaturedependent FRET. We then examined whether ATTO594GM3, the FRET acceptor alone shows temperature-dependent fluorescence in DPPC. Figure 2K and L show emission
spectra of DOPC and DPPC liposomes containing 0.5%
ATTO594-GM3 with excitation at 603 nm at different
temperatures. ATTO594-GM3 did not show temperaturedependent fluorescence both in DOPC and DPPC. This suggests that the number and/or size of fluorescent GM3 analog
clusters were increased at lower temperatures in DPPC.

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Fig. 2  Fluorescent GM3 analogs form clusters in DPPC. A, B Emission spectra of DOPC/C11 TopFluor GM3 (99.5/0.5 mol/mol) (A)or DPPC/C11 TopFluor-GM3 (99.5/0.5 mol/mol) (B) liposomesin the presence and absence of Triton X-100 at 20 °C. Liposomes
were prepared as described in Methods. Excitation at 495 nm.
C, D Emission spectra of DOPC/C11 TopFluor-GM3/ATTO594
GM3 (99/0.5/0.5 mol/mol/mol) (C) or DPPC/C11 TopFluor-GM3/
ATTO594-GM3 (99/0.5/0.5 mol/mol/mol) (D) liposomes in the presence and absence of Triton X-100 at 20 °C. Excitation at 495 nm.
E, F Emission spectra of DOPC/C11 TopFluor GM3/ATTO594
GM3 (99/0.5/0.5 mol/mol/mol) (E) or DPPC/C11 TopFluor-GM3/

ATTO594-GM3 (99/0.5/0.5) (F) liposomes in the presence andabsence of Triton X-100 at 20 ℃. Excitation at 603 nm. G-I Emission
spectra of DOPC/C11 TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5)(G) or DPPC/C11 TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5 mol/
mol/mol) (H, I) liposomes at different temperatures. Excitation at
495 nm. Spectra in H were enlarged in I. J FRET efficiency of (G)and (H) were calculated by Eq. (2). Data are means ± SD of threeexperiments. K, L Emission spectra of DOPC/C11 TopFluor-GM3/
ATTO594-GM3 (99/0.5/0.5 mol/mol/mol) (K) or DPPC/C11 TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5 mol/mol/mol) (L) liposomes
at various temperatures. Excitation at 603 nm

Cell density dependent labeling of cellular GM3
by anti‑GM3 antibody

different temperature dependence. NPA is a genetic defectof acid SMase characterized by the accumulation of SM inlate endosomes/lysosomes [35]. Our results suggest altered
GM3 clustering of the plasma membranes between normal
and NPA fibroblasts.
Previously it was shown that anti-GM3, M2590, labeledsparse but not high density mouse melanoma cells [18]. In
Fig. 3A and B, we labeled sparse and high density MEB4
cells by anti-GM3 antibody, GMR6. The antibody labeled
low density melanoma cells as described in Suppl. Fig. 5.
Similar to M2590, GMR6 antibody did not label high density melanoma cells even at 4 ℃.
Model lipid experiments in Fig. 2 suggest that theobserved difference of anti-GM3 labeling in sparse and confluent melanoma cells may be due to the altered lipid orderin the plasma membranes. To validate this hypothesis, weemployed N-[[4’-N,N-diethylamino-3-hydroxy-6-flavonyl]methyl]-N-methyl-N-(3-sulfopropyl)-1-dodecanaminium,
inner salt (F2N12S) [36] to compare the membrane orderFigure 1 suggests that anti-GM3 antibody labels cellular GM3 in a temperature-dependent manner. In Suppl.
Fig. 5, various cells were fixed and cell surface GM3 waslabeled with anti-GM3 antibody at different temperatures.
It is reported that lipids are not fixed by PFA [34]. All cells
tested were efficiently labeled at 4 ℃. However, labelingwas decreased at higher temperatures. Different cells showed
different labeling temperature thresholds. Human embryonickidney cell line, HEK293, was labeled at 4 ℃ and room
temperature, but not at 37 ℃ and 42 ℃. On the other hand,mouse melanoma cell line, MEB4 was labeled at 37 ℃ but
not at 42 ℃. In Suppl. Fig. 5C, the antibody was incubated at
42 ℃ for 30 min before labeling MEB4 cells at 4 ℃. Suppl.
Fig. 5C indicates that the antibody was not inactivated at
42 ℃. Interestingly, normal human skin fibroblast and skinfibroblast from Niemann-Pick A (NPA) patient showed

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Fig. 3  Cell density dependent labeling of cellular GM3 by antiGM3 antibody. A Low density (1.4 ± 0.4 × ­104 cells/15 mmϕ coverslip (n = 3)) and high density (2.8 ± 0.6 × ­105 cells/15 mmϕ coverslip(n = 4)) MEB4 cells were fixed and labeled with anti-GM3 antibody
as described in Methods. Cells were doubly labeled with phalloidin to
identify cells. B Distribution of cells with different labeling at different temperatures. #1, whole cells were labeled; #2, only a part of cells
were labeled. #3, no labeling. The numbers on the top of the bars arenumber of cells counted. C FLIM images of low density and highdensity MEB4 cells labeled with F2N12S at 25 ℃. Cells were labeled
as described in Methods. The pixel colors describe the lifetime values in picosecond according to the color scale on the Y axis. D Lifetime distribution for low and high density cells. The data in the table
are the mean and standard deviation of the average lifetime obtainedwith the 12 images taken individually. Two-photon excitation was
at 830 nm and fluorescence was collected through a band-pass filter
585 nmbetween sparse and confluent MEB4. Being conjugated to azwitter ion headgroup and a long alkyl chain, the two-color
F2N12S fluorophore is selectively anchored at the plasma
membrane outer leaflet [36]. Our previous data showed thatthe long-lived and mean lifetimes of the tautomer (T*) bandof F2N12S differ by a factor of 2 between liquid-ordered(Lo) and liquid-disordered (Ld) phases, and thus can beused by fluorescence lifetime imaging microscopy (FLIM)
to sensitively and straightforwardly monitor the changes
in lipid phase in cells [37]. Figure 3C and D compare the
FLIM images of low density and high density MEB4 cells.
FLIM of low density cells showed homogeneous distributionof lifetimes, exhibiting a mean lifetime value 5.6 ± 0.4 ns.

This value was significantly decreased in high density cells(4.0 ± 1.0 ns), indicating a decrease in the lipid order in confluent cells.

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Lipidomics indicates a different compositionof phospholipids in sparse and confluent melanoma
cells
Since F2N12S selectively labels the outer leaflet of theplasma membrane, the decrease of lipid order in highdensity MEB4 cells suggests that the lipid compositionof the outer leaflet may differ between high density andlow density cells. Lipid order of the outer leaflet of theCell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	Page 7 of 15

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Fig. 4  Different composition of phospholipid molecular species insparse and confluent melanoma cells. A 2D-HPTLC analysis of lipid
composition of low density and high density MEB4 cells. 2D-HPTLC
was performed as described in Methods. B Cell surface labeling of
low density and high density cells with mCherry-D4. C, D Lipidomics analyses of phosphatidylcholine (PC) (C) and sphingomyelin(SM) (D) from low density and high density cells. Lipidomics was
performed as described in Methods. Data in C and D are mean ± SDof three experiments. *P < 0.05, **P < 0.01. (E) Anti-GM3 antibody
labeling of low density MEB4 cells treated with oleic acid. Oleic acidtreatment was performed as described in Methods

plasma membrane is sensitive to the content of cholesterol, sphingolipids, unsaturated fatty acids and saturated
fatty acids. Figure 4A shows the results of 2D-HPTLC
analysis of lipids extracted from low density and high density MEB4 cells. Orcinol labeling revealed glycolipids,
whereas cupric acetate labeled all lipids. Figure 4A indicates that gross lipid composition was not significantly different between low density and high density cells. This is
supported by the observation that cell surface cholesterolrich domains labeled with mCherry-D4 [38, 39] did not
significantly differ between low density and high density
cells (Fig. 4B).
We then examined different molecular species ofphospholipids by lipidomics (Fig. 4C, D and Suppl. Figure 6). There was no significant difference in content andmolecular species of sphingomyelin (SM) between low
density and high density cells (Fig. 4D). In contrast, significant decrease of DPPC (32:0 PC), increase of several
mono- and di-unsaturated molecular species (30:1 PC,
O-32:1 PC, O-34:2 PC and O-36:2 PC) and decrease ofpolyunsaturated PCs were observed (Fig. 4C). Increase ofmono- or di-unsaturated molecular species and decreaseof polyunsaturated molecular species in high density cells
were also observed in phosphatidylethanolamine and phosphatidylinositol (Suppl Fig. 6). In phosphatidylserine,
both saturated and unsaturated molecular species weredecreased in high density cells (Suppl Fig. 6).
Phospholipids are asymmetrically distributed in theplasma membrane [25, 40, 41], the outer leaflet of theplasma membrane is enriched with phosphatidylcholine

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and sphingomyelin. Since there were no significant differences in sphingomyelin molecular species between
low density and high density melanoma cells, sphingomyelin may not contribute to the observed alteration oflipid order in high density cells. Fatty acid compositionof phosphatidylcholine in the outer leaflet of nucleatedcells is not fully understood. However, analysis of lipid
composition of envelope viruses bud from different plasma
membrane domains suggests the enrichment of monounsaturated phosphatidylcholine in the plasma membraneof human skin fibroblasts [42]. Our results showed theincrease of monounsaturated phosphatidylcholine in highdensity cells, suggesting that the increase of monounsaturated phosphatidylcholine is responsible for the decreased
lipid order in high density cells.
We then examined whether the addition of monounsaturated fatty acid, oleic acid (C18:1) to low density cells
mimic high density cells. Exogenously added oleic acid israpidly incorporated to cells [43]. Oleic acid stimulates
lipid droplet formation by activating the long-chain fatty
acid receptor FFAR4 [44]. Results of fluorescent fatty acidanalogs suggest that the exogenously added fatty acids areaccumulated to lipid droplets [45, 46] in which neutral
lipids, triglyceride and cholesterol ester are enriched. In
addition to neutral lipids, oleic acid is incorporated into
phospholipids. In HepG2 cells, oleic acid is mainly incorporated to phosphatidylcholine (8 nmol/mg protein after2 h) and triglyceride (6 nmol/mg) followed by phosphatidylethanolamine (3 nmol/mg) [47]. Efficient incorporation of oleic acid to phosphatidylcholine is reported inother cell types [48–51]. Anti-GM3 labeling became very
dim after 2 h incubation of low density cells with oleic
acid (Fig. 4E). These results suggest that an alteration of
fatty acid composition in high density cells is the cause of
decreased anti-GM3 labeling and the differences in GM3
microenvironment as compared to low density melanoma
cells.

Discussion
Partner lipid‑dependent binding to GM3
is a common property of anti‑GM3 antibodies
In this study, we examined binding of three independentlyisolated anti-GM3 monoclonal antibodies to DOPC/GM3
and DPPC/GM3. All three antibodies showed preference to
DPPC/GM3, suggesting that partner lipid-dependent binding
to GM3 is a common characteristic of anti-GM3 antibodies. The binding of GMR6 antibody to GM3 in ELISA wasdependent on the gel-to-liquid crystalline phase transition
temperature of partner lipids and incubation temperature,

13

M. Murate et al.

showing higher binding in gel phase lipids than in liquid
crystalline phase lipids. In contrast, the binding to GM3
alone was not affected by incubation temperature. The binding of GMR6 antibody is dependent on the chain lengths of
the fatty acyl chain of GM3 and the antibody did not significantly bind to lyso GM3. These results suggest that theantibody recognizes both hydrophilic headgroup and hydrophobic ceramide moiety of GM3. Since GM3 forms clusters
in DPPC, our results suggest that the epitope of the antibody
may be both aggregated sugar and ceramide moiety of GM3.

GM3 is clustered in DPPC
Our liposome experiments with fluorescent GM3 analogs
indicate that GM3 analogs were highly aggregated in DPPC.
Thus, our results suggest that anti-GM3 antibodies bindclustered GM3. Aggregation of GM3 in DPPC is consistent with previous small-angle X-ray diffraction observationthat GM3 and DPPC are phase separated [52, 53]. Whereas
more complicated gangliosides form micelles, GM3 forms
bilayers. The main phase transition of GM3 is reported to
be 35–40 ℃ [26, 54]. This is very close to the phase transition temperature of DPPC (­ Tm = 41 ℃) [26]. In contrast toGM3, palmitoyl (C16:0) SM (pSM) (­ Tm = 40 ℃) [26] forms
clusters in DOPC and is well mixed with DPPC [55]. In
contrast to pSM, main fatty acids in milk GM3 are C22:0
22%, C23:0 34% and C24:0 20% (manufacturer’s data sheet).
It is speculated that GM3 did not mix with DPPC due tothe difference of the hydrophobic acyl chains. However, thisis unlikely since the antibody binds preferentially synthetic
C16:0 GM3/DPPC to C16:0 GM3/POPC, suggesting that
C16:0 GM3 also phase separated from DPPC. Since pSM
and C16:0 GM3 share hydrophobic ceramide moiety, hydrophilic carbohydrate moiety of GM3 may be involved in the
different interaction of pSM and GM3 to DOPC and DPPC.
Atomistic MD simulations of DOPC/Chol/SM (45/25/10)
bilayers containing 20 mol % GM3 showed that GM3
interacted most strongly with themselves, followed by SM,
DOPC and Chol [56]. Interestingly, 65% of the interactionenergy of between GM3 is generated by interactions between
headgroups [56].
Our results indicate that GM3 clusters were preferentiallyformed in less fluid membrane or at low temperature. Carbohydrate to carbohydrate interaction of GM3 may occur bothin solid and fluid membranes. GM3 is reported to spontaneously transfer between membranes above ­Tm [57]. We speculate that spontaneous detachment and insertion of GM3 into
DOPC-rich fluid regions of the membrane disturbs the stableformation of GM3 clusters (Fig. 5).
In contrast to GM1-cholera toxin interaction [17], thepresence of cholesterol did not affect the binding of antiGM3 antibody, GMR6, suggesting that cholesterol does notchange the conformation of GM3. This may be due to theCell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	Page 9 of 15

167

Fig. 5  Putative distributionof GM3 in DOPC and DPPC.
Hydrogen bond between
glycans facilitates clusteringof GM3 both in DOPC andDPPC membrane. In DOPC,
GM3 readily detaches from themembrane and then inserts intoGM3-poor fluid DOPC-richarea. GM3 clusters are morestable in DPPC

relatively small headgroup of GM3 compared to GM1. Thisobservation may be related to the MD simulation results [56]that GM1 and GM2 nanodomains were enlarged by cholesterol whereas the domain size of GM3 was not affected due
to strong GM3-GM3 interaction.

Cell density dependent labeling of cellular GM3
by anti‑GM3 antibody
Similar to ELISA results, the binding of anti-GM3 to cellsurface GM3 showed temperature dependence. While cells
were efficiently labeled at 4 ℃, labeling was less efficientat higher temperatures. These results suggest that the antiGM3 antibody preferentially binds to GM3 in less fluid lipid
environment.
It has long been known that in several cell types, antiGM3 antibody labels sparse but not confluent cells [18, 19].
Since cell density does not affect GM3 content and GM3 canbe chemically labeled in a cell-density-independent manner[18, 19], it is speculated that GM3 is cryptic in confluent
cells. However, the molecular mechanism of the crypticity is
not known. Confluent melanoma cells were not labeled withthe antibody even at 4 ℃, suggesting strong differences inlipid distribution between sparse and confluent cells. Indeed,
our FLIM experiments showed huge difference in lipid orderin low density and high density cells.
Anti-GM3 antibodies added to the medium only labels
the outer leaflet of the plasma membrane. Phospholipids areasymmetrically distributed in the plasma membrane [40] andthe outer leaflet is enriched with sphingomyelin (88% in theouter leaflet in human skin fibroblasts) and phosphatidylcholine (92% in the outer leaflet) [25]. In contrast, more than
95% of phosphatidylethanolamine, phosphatidylinositol andphosphatidylserine are localized in the inner leaflet [25].
Our results using outer leaflet specific dye, F2N12S, indicate the alteration of physical properties of sphingomyelin
and phosphatidylcholine-enriched outer leaflet of the plasma
membrane between low and high density cells. Biochemical

analysis of lipids indicates that the gross lipid composition and cholesterol content were not significantly altered
between low density and high density cells. Cholesterolbinding D4 labeling suggests that cell surface cholesterol
distribution is also similar between high and low density
cells. Content and fatty acid composition of sphingomyelin
were also not affected. In contrast, there were differencesin phosphatidylcholine molecular species between low andhigh density cells. There were a decrease in DPPC andincreases in mono- and di-unsaturated molecular species
whereas polyunsaturated molecular species were decreasedin high density cells. Although the molecular species ofphospholipids in the outer and inner leaflet of red blood cells
have been studied [41], lipidomics of the outer and innerleaflet of the plasma membrane from nucleated cells has
not been reported. Plasma membrane phospholipids were
estimated to be 13% of total cellular phospholipids in baby
hamster kidney (BHK) cells [58]. Using different enveloped
viruses, the enrichment of monounsaturated- and decreaseof polyunsaturated-phosphatidylcholine in the plasma
membrane in human skin fibroblasts are suggested [42].
Our results showed that the addition of oleic acid (C18:1)
to low density cells abolished anti-GM3 labeling. Oleic acidis reported to be efficiently incorporated to phosphatidylcholine [51]. Our results suggest the role of monounsaturated
molecular species of phosphatidylcholine in alteration ofGM3 microenvironment.
It is reported that lipids are mobile even after paraformaldehyde fixation [34]. Thus we cannot inhibit the formationof pseudo-cluster of GM3 by multivalent IgM antibodies.
However, our results indicate that the pre-existed lipid cluster is required for the binding of the antibody. In summary,our results suggested that anti-GM3 antibody bind to GM3
only when GM3 forms clusters. Formation of clusters isdependent on fatty acid composition of partner lipids bothin model and cell membranes. Our results revealed that thecrypticity of GM3 against anti-GM3 antibody is due to the

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M. Murate et al.

alteration of fatty acid composition of the surrounding lipids
of GM3.

Enzyme‑linked immunosorbent assay (ELISA)

Methods

Surface plasmon resonance (SPR)

Antibodies

GM3-containing liposomes were prepared as follows:
Fig. 1D: 50 nmol of GM3 in a chloroform/methanol (2:1)
solution was mixed with either 250 nmol DOPC or DPPCin the same solvent and dried under nitrogen. Figure 1E, thesolutions containing 250 nmol DPPC and 100 nmol GM3
with or without 250 nmol cholesterol were prepared. In
Fig. 1F, 25 nmol of GM3 was mixed with 225 nmol DPPC.
Dried samples were mixed vigorously in 200 μl of HBS-N
buffer (10 mM Hepes, pH 7.4, 150 mM NaCl). GM3 containing liposomes were prepared by sonicating 5 min threetimes in bath sonicator. The initial temperature of the water
bath was set at 41 ℃ (Fig. 1D and E) or room temperature (Fig. 1F). Alternatively, liposomes were frozen in liquid nitrogen and thawed at 50 ℃. This cycle was repeatedthree times. All conditions gave similar results. Liposome
solutions were filtered through a 0.22 μmϕ polyvinylidene
difluoride (PVDF, Millipore Co., Burlington, MA). These
solutions were diluted ten times with HBS-N buffer, thenimmobilized on a biosensor L1 chip (Cytiba, Tokyo, Japan)
at flow rate 10 μL/min. The chip surface was washed with
50 mM NaOH, then blocked with 100 μg/mL BSA in HBS-N
buffer. Experiments were carried out at 25 ℃ at flow rate 10
μL/min (Fig. 1D and E) or 30 μL/min (Fig. 1F). The binding of the anti-GM3 antibody to GM3 was analyzed with a
Biacore 3000 analytical system. Data were evaluated with
the BIAevaluation 4.1.1 software program (Biacore, Cytiba).
The results shown represent 3–4 independent experiments.

Anti-GM3 monoclonal IgM clones GMR6 and M2590
were obtained from TCI Europe (Zwijndrecht, Belgium)
and Cosmo Bio (Tokyo, Japan), respectively. Anti-GM3
IgG clone DH2 was obtained as described [8]. HRP-labeledanti-mouse IgM was from Sigma (St Louis, MI). HRPlabeled anti-mouse IgG was from GE Healthcare (Chicago,
Il). Alexa488-labeled anti-mouse IgM was from Molecular
probes (Eugene, OR).

Lipids and other reagents
Glucosylceramide (Gaucher’s spleen, GlcCer), lactosylceramide (bovine buttermilk, LacCer), N-acyl-sphingosylphosphorylethanolamine (bovine buttermilk, CerPE), monosialogangliosides, GM1 (Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1’-ceramide) from bovine and GM3(Neu5Acα2,3 Galβ1,4Glcβ1,1’-ceramide) from bovine
buttermilk were from Matreya LLC (State College, PA).
Major fatty acids of buttermilk GM3 were C16:0 6%,
C22:0 22%, C23:0 34% and C24:0 20% according to the
manufacturer. C8:0, C12:0, C16:0 and C24:0-GM3 werefrom Nagara Science (Gifu, Japan). The following lipids
were purchased from Avanti Polar Lipids (Alabaster, AL):
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
palmitoyl sphingomyelin (pSM), ceramide (porcine brain,
Cer), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine(DOPS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-racglycerol) (DOPG), 1',3'-bis[1,2-dioleoyl-sn-glycero3-phospho]-glycerol (TOCL), C11 TopFluor-GM3. GM2(GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1’-ceramide)
from bovine brain was from Wako Pure Chemical Industries (Osaka, Japan). Lyso GM3 was provided by Sonnino’s
laboratory and prepared as described [59]. ATTO594-GM3
was prepared as described [28]. N-[[4’-N,N-diethylamino3-hydroxy-6-flavonyl]-methyl]-N-methyl-N-(3-sulfopropyl)1-dodecanaminium, inner salt (F2N12S) was prepared as
described [36]. mCherry-D4 was prepared as described [60].

ELISA was performed as described previously [61].

Fluorescence measurement of liposomes
Small unilamellar vesicles (SUVs) containing DPPC/C11
TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5), DOPC/
C11 TopFluor-GM3/ATTO594-GM3 (99/0.5/0.5), DPPC/ATTO594-GM3 (99.5/0.5), DOPC/ATTO594-GM3(99.5/0.5) were prepared in PBS by ethanol injection [62,63]. Fluorescence spectra measurements were recorded witha Fluoromax Plus spectrofluorometer (Horiba Jobin–Yvon,
Lille, France) equipped with a thermostat cell compartment.
To solubilize lipids, Triton X-100 (final concentration 1%)
was added. FRET efficiency was calculated as()(Emission of donor F507 in the absence of acceptor()− Emission of donor F507 in the presence of acceptor)()∕Emission of donor F507 in the absence of acceptor
(1)
or

13

Cell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	()()(Emission of donor F507 + Emission of acceptor F626 )()∕Emission of donor F507
(2)

Cells
Human embryonic kidney cell line, HEK293 was grownin Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% L-glutamine, 100
units/mL penicillin and 100 µg/mL streptomycin. Mousemelanoma cell line, MEB4 was cultured as described [64].
Human skin fibroblasts were grown as described [65]. Niemann-Pick A fibroblasts were grown as described [66].

Antibody labeling of cells

Page 11 of 15

167

Fluorescence lifetime imaging microscopy (FLIM)
Sparse and confluent MEB4 cells were washed with PBS
and then stained by adding a freshly prepared solution of
F2N12S in PBS to a final concentration of 0.1 µM per 1­ 06
cells (< 0.25% DMSO volume), and incubated for 2 minin the dark at room temperature [37]. The cells were thenwashed again with PBS before measurement. We performed
two-photon fluorescence microscopy at 25 ℃ by using an
in-house-built two-photon laser scanning setup based on
an inverted microscope (IX70, Olympus, Tokyo, Japan)
with an 60 × 1.2NA water immersion objective (UPlanApo, Olympus) and two fast galvo mirrors in the descanned
fluorescence collection mode [67, 68]. Two-photon excitation was provided by a femtosecond laser (Insight DeepSee, Spectra Physics, Milpitas, CA). The typical excitation
power was ~ 2.5 mW (λ = 830 nm) at the sample. Photons
were detected using an avalanche photodiode (SPCM-AQR14-FC; Perkin Elmer) coupled to an HQ 585/40 bandpass
filter and a single photon-counting TCSPC module (SPC830,
Becker & Hickl, Berlin, Germany) operating in the reversed
start-stop mode. Acquisition times were adjusted to achieve
1000 photons per pixel. The minimum fluorescence lifetime
detectable with this setup is ~ 300 ps.
FLIM data were analyzed using the FLIMfit software(Imprerial College London) [69]. The images were initiallysegmented to analyze only data from plasma membranes.
The lifetimes were recovered from the fluorescence decay
using an iterative reconvolution method [70]. The goodnessof the fit was evaluated from the χ2 values, which ranged
from 0.9 to 1.2, and from the plot of the distribution of theresiduals.

HEK293 cells were grown on a plastic bottom dish (Ibidi,
Gräfelfing, Germany), and MEB4 cells, human skin fibroblasts, and NPA fibroblasts were grown on glass coverslips
to 30–40% confluent and fixed with 4% paraformaldehyde
in PBS for 20 min at room temperature. Cells were thenblocked with 3% BSA in PBS for 1 h, pre-warmed for 20 min
at indicated temperatures, and labeled with 22.4 µg/ml antiGM3 antibody, GMR6, for 1 h at indicated temperatures, followed by incubation with Alexa488-labeled anti-mouse IgM
secondary antibody for 1 h. After washing with PBS, thecoverslips were mounted with ProLong Diamond antifade
mountant (Invitrogen, Carlsbad, CA). The specimens wereobserved under an LSM700 confocal microscope (Zeiss,
Oberkochen, Germany).
To obtain low and high densities MEB4 cell line, cells
were seeded on the glass coverslips at 30–40% in low density
and 90–120% in high density. After culturing overnight, celldensities were examined under a light microscope (Nikon,
Tokyo, Japan). Cells were fixed with 4% paraformaldehyde
in PBS for 20 min at room temperature, blocked with 3%
BSA in PBS for 1 h, pre-warmed for 20 min at different
temperatures, and then labeled with 22.4 µg/ml anti-GM3
antibody for 1 h at indicated temperatures, followed byincubation with Alexa488-labeled anti-mouse IgM secondary antibody for 1 h. After washing with PBS, actin fibers
were stained with Alexa594-labeled phalloidin (Molecular
Probes) as described [18]. The coverslips were mounted with
ProLong Diamond antifade mountant. The specimens wereobserved under an LSM700 confocal microscope.

MEB4 cells were grown in glass-bottom dish to low density.
The medium was exchanged to serum-free medium containing 0.4% BSA and 500 µM oleic acid. After 1 h or 2 h incubation at 37 ℃, cells were fixed and labeled with anti-GM3
antibody as described above.

Confocal microscopy

Lipid analyses

Stained cells were imaged using an LSM700 confocal microscope equipped with a C-Apochromat 63XW Korr (1.2 NA)
objective.

The same numbers of MEB4 cells were cultured in 15 cm(low density) and 3.5 cm (high density) dishes, respectively.
Cells were washed three times with ice-cold PBS, scraped,
washed three times again in ice-cold PBS, and divided to one

mCherry‑D4 labelingLow density and high density MEB4 cells were prepared
as described above. Cell surface mCherry-D4 labeling was
performed as described [71].

Oleic acid treatment of low density MEB4 cells

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Page 12 of 15

portion for protein determination and nine portions for lipid
extraction. For protein determination, cells were collected bycentrifugation, lysed by vortex in 0.1% Triton X-100, 2 mM
EDTA in Milli Q (pH 7.4), and fragmented using probe sonicator for 1 min, followed by freeze-thawing. Protein contents
were measured using a BCA protein assay kit (ThermoFisher
Scientific, Waltham, MA). For lipid extraction, cells werecollected by centrifugation and extracted lipids with chloroform/methanol (1:2 by vol., and then 2:1 by vol.). Lipids
were applied to HPTLC plates (Merck, Darmstadt, Germany) by adjusting with protein contents. The plates weredeveloped sequentially in (1) chloroform/methanol/formic
acid/water (65:25:8.9:1.1 by vol.), (2) chloroform/methanol/4.4 M ammonia (50:40:10 by vol.), and (3) diethylether[39, 72]. Glycolipids were visualized by spraying orcinol
reagent and heating at 100 ℃ while phospholipids and neutral lipids were visualized by spraying cupric acetate solution to the same plates and heating at 180 ℃. The position ofeach lipid on HPTLC was determined using lipid standards.
For the detection of phospholipid molecular species,
cells were prepared as described above and LC/ESI–MSbased lipidomics analyses were performed on a Shimadzu
Nexera UPLC system (Shimadzu, Kyoto, Japan) coupledwith a QTRAP 4500 hybrid triple quadrupole linear ion
trap mass spectrometer (AB Sciex, Framingham, MA).
Lipids extracted from melanoma cells [73] were injectedby an auto sampler. Chromatographic separation was performed on a SeQuant ZIC-HILIC PEEK coated column(250 mm × 2.1 mm, 1.8 µm; Millipore) maintained at 50 ℃
using mobile phase A (water/acetonitrile (95/5, v/v) containing 10 mM ammonium acetate) and mobile phase B (water/acetonitrile (50/50, v/v) containing 20 mM ammonium acetate) in a gradient programme (0–22 min: 0% B → 40% B;
22–25 min: 40% B → 40% B; 25–30 min: 0% B) with a flowrate of 0.5 mL/min. The instrument parameters for negative
ion mode were as follows: curtain gas, 10 psi; collision gas,
7 arb. unit; ionspray voltage, − 4500 V; temperature, 700 ℃;
ion source gas 1, 30 psi; ion source gas 2, 70 psi. Detectionof phospholipid species was performed by multiple reaction
monitoring (MRM) as described in Supplemental Table 1.
Supplementary Information The online version contains supplementary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00018-0​ 23-0​ 4813-9.
Author contributions Conceptualization, TK; methodology, LR, NH
and JA; investigation, MM, NY, NT, LR, BP, AM and NK; resources
LM, SS, NK, HA, MKK, YK, KI and JI; writing—original draft, MM
and TK; writing—review and editing, MM, NT, LR, NH, NK, JA, YS,
SS, JI, YM, KI and TK; supervision, JI, KI, YM and TK.
Funding Mizutani Foundation for Glycoscience (to N.Y. and T.K).
RIKEN Glycolipidologue Initiative Program (to T.K. and Y.S.). Vaincre les Maladies Lysosomales (19/LBPH/S44 to T.K.) Japan Agency
for Medical Reasearch and Development (AMED) (JP22ama121008
and JP22am0401013 to Y.K.) Agence Nationale pour la Recherche(ANR-19-CE16-0012-02 and ANR-22-CE44-0019-01 to T.K.). ITMO

13

M. Murate et al.
Cancer of Aviesan within the framework of the 2021–2030 Cancer
Control Strategy, on funds administered by Inserm (to T.K.). Y.M. isgrateful to the Institut Universitaire de France (IUF) for financial support and providing additional time to be dedicated to research.
Data and materials availability The datasets generated and/or analyzed
during the current study are available from the corresponding author
on reasonable request.

Declarations
Conflict of interest Authors declare that they have no competing interests.
Ethical approval and consent to participate Not applicable.
Consent for publication All authors agreed on the final version of themanuscript.

References
1. Zheng M, Fang H, Tsuruoka T, Tsuji T, Sasaki T, Hakomori S(1993) Regulatory role of GM3 ganglioside in alpha 5 beta 1
integrin receptor for fibronectin-mediated adhesion of FUA169
cells. J Biol Chem 268(3):2217–2222. https://​doi.​org/​10.​1016/​
S0021-​9258(18)​53984-3
2. Kabayama K, Sato T, Saito K, Loberto N, Prinetti A, Sonnino S,
Kinjo M, Igarashi Y, Inokuchi J (2007) Dissociation of the insulin
receptor and caveolin-1 complex by ganglioside GM3 in the stateof insulin resistance. Proc Natl Acad Sci USA 104(34):13678–
13683. https://​doi.​org/​10.​1073/​pnas.​07036​50104
3. Coskun U, Grzybek M, Drechsel D, Simons K (2011) Regulation of human EGF receptor by lipids. Proc Natl Acad Sci USA
108(22):9044–9048. https://​doi.​org/​10.​1073/​pnas.​11056​66108
4. Nakano M, Hanashima S, Hara T, Kabayama K, Asahina Y, Hojo
H, Komura N, Ando H, Nyholm TKM, Slotte JP, Murata M (2021)
FRET detects lateral interaction between transmembrane domainof EGF receptor and ganglioside GM3 in lipid bilayers. Biochim
Biophys Acta 1863:183623. https://​doi.​org/​10.​1016/j.​bbamem.​
2021.​183623
5. Inokuchi JI, Kanoh H, Inamori KI, Nagafuku M, Nitta T, Fukase
K (2021) Homeostatic and pathogenic roles of the GM3 ganglioside. FEBS J 289:5152–5165. https://​doi.​org/​10.​1111/​febs.​16076
6. Hirabayashi Y, Hamaoka A, Matsumoto M, Matsubara T, Tagawa
M, Wakabayashi S, Taniguchi M (1985) Syngeneic monoclonal
antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma. J Biol Chem 260(24):13328–13333. https://​doi.​org/​10.​1016/​S0021-​9258(17)​38873-7
7. Kotani M, Ozawa H, Kawashima I, Ando S, Tai T (1992) Generation of one set of monoclonal antibodies specific for a-pathway
ganglio-series gangliosides. Biochim Biophys Acta 1117(1):97–
103. https://​doi.​org/​10.​1016/​0304-​4165(92)​90168-t
8. Dohi T, Nores G, Hakomori S (1988) An IgG3 monoclonal antibody established after immunization with GM3 lactone: immunochemical specificity and inhibition of melanoma cell growthin vitro and in vivo. Cancer Res 48:5680–5685
9. Gomez-Mouton C, Abad JL, Mira E, Lacalle RA, Gallardo E,
Jimenez-Baranda S, Illa I, Bernad A, Manes S, Martinez AC(2001) Segregation of leading-edge and uropod components into
specific lipid rafts during T cell polarization. Proc Natl Acad Sci
USA 98:9642–9647. https://​doi.​org/​10.​1073/​pnas.​17116​0298

Cell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	
10. Janich P, Corbeil D (2007) GM1 and GM3 gangliosides highlight
distinct lipid microdomains within the apical domain of epithelial
cells. FEBS Lett 581:1783–1787. https://​doi.​org/​10.​1016/j.​febsl​
et.​2007.​03.​065
11. Fujita A, Cheng J, Hirakawa M, Furukawa K, Kusunoki S, Fujimoto T (2007) Gangliosides GM1 and GM3 in the living cell
membrane form clusters susceptible to cholesterol depletion andchilling. Mol Biol Cell 18:2112–2122. https://​doi.​org/​10.​1091/​
mbc.​e07-​01-​00712
12. Chigorno V, Palestini P, Sciannamblo M, Dolo V, Pavan A, Tettamanti G, Sonnino S (2000) Evidence that ganglioside enricheddomains are distinct from caveolae in MDCK II and human fibroblast cells in culture. Eur J Biochem 267(13):4187–4197. https://​doi.​org/​10.​1046/j.​1432-​1327.​2000.​01454.x
13. Cutillo G, Saariaho AH, Meri S (2020) Physiology of gangliosides and the role of antiganglioside antibodies in human diseases. Cell Mol Immunol 17:313–322. https://​doi.​org/​10.​1038/​
s41423-​020-​0388-9
14. Willison HJ, Yuki N (2002) Peripheral neuropathies and antiglycolipid antibodies. Brain 125:2591–2625. https://​doi.​org/​10.​1093/​brain/​awf272
15. Lloyd KO, Gordon CM, Thampoe IJ, DiBenedetto C (1992) Cellsurface accessibility of individual gangliosides in malignant melanoma cells to antibodies is influenced by the total gangliosidecomposition of the cells. Cancer Res 52:4948–4953
16. Greenshields KN, Halstead SK, Zitman FM, Rinaldi S, Brennan
KM, O’Leary C, Chamberlain LH, Easton A, Roxburgh J, PedianiJ, Furukawa K, Furukawa K, Goodyear CS, Plomp JJ, Willison
HJ (2009) The neuropathic potential of anti-GM1 autoantibodies
is regulated by the local glycolipid environment in mice. J Clin
Invest 119:595–610. https://​doi.​org/​10.​1172/​JCI37​338
17. Lingwood D, Binnington B, Rog T, Vattulainen I, Grzybek M,
Coskun U, Lingwood CA, Simons K (2011) Cholesterol modulates glycolipid conformation and receptor activity. Nat Chem Biol
7:260–262. https://​doi.​org/​10.​1038/​nchem​bio.​551
18. Sakiyama H, Takahashi T, Hirabayashi Y, Taniguchi M (1987)
Change in the topographical distribution of GM3 during cellspreading and growth: immunostaining with monoclonal antibody against GM3. Cell Struct Funct 12:93–105. https://​doi.​org/​
10.​1247/​csf.​12.​93
19. Tatewaki K, Yamaki T, Maeda Y, Tobioka H, Piao H, Yu H,
Ibayashi Y, Sawada N, Hashi K (1997) Cell density regulates
crypticity of GM3 ganglioside on human glioma cells. Exp CellRes 233:145–154. https://​doi.​org/​10.​1006/​excr.​1997.​3563
20. Kotani M, Kawashima I, Ozawa H, Ogura K, Ishizuka I,
Terashima T, Tai T (1994) Immunohistochemical localization ofminor gangliosides in the rat central nervous system. Glycobiology 4:855–865. https://​doi.​org/​10.​1093/​glycob/​4.6.​855
21. Sorice M, Parolini I, Sansolini T, Garofalo T, Dolo V, Sargiacomo
M, Tai T, Peschle C, Torrisi MR, Pavan A (1997) Evidence for the
existence of ganglioside-enriched plasma membrane domains inhuman peripheral lymphocytes. J Lipid Res 38:969–980. https://​doi.​org/​10.​1016/​S0022-​2275(20)​37221-7
22. Misasi R, Sorice M, Garofalo T, Griggi T, Campana WM, Giammatteo M, Pavan A, Hiraiwa M, Pontieri GM, O’Brien JS (1998)
Colocalization and complex formation between prosaposinand monosialoganglioside GM3 in neural cells. J Neurochem
71:2313–2321. https://​doi.​org/​10.​1046/j.​1471-​4159.​1998.​71062​
313.x
23. Garofalo T, Lenti L, Longo A, Misasi R, Mattei V, Pontieri GM,
Pavan A, Sorice M (2002) Association of GM3 with Zap-70
induced by T cell activation in plasma membrane microdomains:
GM3 as a marker of microdomains in human lymphocytes. J Biol
Chem 277:11233–11238. https://​doi.​org/​10.​1074/​jbc.​M1096​
01200

Page 13 of 15167

24. Chen Y, Qin J, Chen ZW (2008) Fluorescence-topographic NSOM
directly visualizes peak-valley polarities of GM1/GM3 rafts in cell
membrane fluctuations. J Lipid Res 49:2268–2275. https://d​ oi.o​ rg/​
10.​1194/​jlr.​D8000​31-​JLR200
25. Murate M, Abe M, Kasahara K, Iwabuchi K, Umeda M, Kobayashi T (2015) Transbilayer distribution of lipids at nano scale.
J Cell Sci 128:1627–1638. https://​doi.​org/​10.​1242/​jcs.​163105
26. Marsh D (2013) Handbook of lipid bilayers, 2nd edn. CRC Press,
Boca Raton
27. Taniguchi M, Wakabayashi S (1984) Shared antigenic determinant expressed on various mammalian melanoma cells. Gan
75:418–426
28. Komura N, Suzuki KG, Ando H, Konishi M, Koikeda M, Imamura A, Chadda R, Fujiwara TK, Tsuboi H, Sheng R, Cho W,
Furukawa K, Furukawa K, Yamauchi Y, Ishida H, Kusumi A,
Kiso M (2016) Raft-based interactions of gangliosides with a GPIanchored receptor. Nat Chem Biol 12:402–410. https://d​ oi.o​ rg/1​ 0.​1038/​nchem​bio.​2059
29. MacDonald RI (1990) Characteristics of self-quenching of thefluorescence of lipid-conjugated rhodamine in membranes. J Biol
Chem 265:13533–13539. https://d​ oi.o​ rg/1​ 0.1​ 016/S​ 0021-9​ 258(18)​77380-​77388
30. Elvington SM, Nichols JW (2007) Spontaneous, intervesiculartransfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs. Biochim Biophys Acta 1768:502–508. https://​doi.​
org/​10.​1016/j.​bbamem.​2006.​11.​013
31. Coste V, Puff N, Lockau D, Quinn PJ, Angelova MI (2006) Raftlike domain formation in large unilamellar vesicles probed by
the fluorescent phospholipid analogue, C12NBD-PC. Biochim
Biophys Acta 1758:460–467. https://​doi.​org/​10.​1016/j.​bbamem.​2006.​03.​003
32. Puff N, Watanabe C, Seigneuret M, Angelova MI, Staneva G(2014) Lo/Ld phase coexistence modulation induced by GM1.
Biochim Biophys Acta 1838:2105–2114. https://​d oi.​o rg/​1 0.​1016/j.​bbamem.​2014.​05.​002
33. Xu X, London E (2000) The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid
domain formation. Biochemistry 39(5):843–849. https://​doi.​org/​
10.​1021/​bi992​543v
34. Tanaka KA, Suzuki KG, Shirai YM, Shibutani ST, Miyahara
MS, Tsuboi H, Yahara M, Yoshimura A, Mayor S, Fujiwara
TK, Kusumi A (2010) Membrane molecules mobile even after
chemical fixation. Nat Methods 7:865–866. https://​doi.​org/​10.​1038/​nmeth.f.​314
35. Brady RO, Kanfer JN, Mock MB, Fredrickson DS (1966) The
metabolism of sphingomyelin. II. Evidence of an enzymatic
deficiency in Niemann-Pick diseaese. Proc Natl Acad Sci USA
55:366–369. https://​doi.​org/​10.​1073/​pnas.​55.2.​366
36. Shynkar VV, Klymchenko AS, Kunzelmann C, Duportail G,
Muller CD, Demchenko AP, Freyssinet JM, Mely Y (2007)
Fluorescent biomembrane probe for ratiometric detection of
apoptosis. J Am Chem Soc 129(7):2187–2193. https://​doi.​org/​
10.​1021/​ja068​008h
37. Kilin V, Glushonkov O, Herdly L, Klymchenko A, Richert L,
Mely Y (2015) Fluorescence lifetime imaging of membrane
lipid order with a ratiometric fluorescent probe. Biophys J
108:2521–2531. https://​doi.​org/​10.​1016/j.​bpj.​2015.​04.​003
38. Shimada Y, Maruya M, Iwashita S, Ohno-Iwashita Y (2002) The
C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains.
Eur J Biochem 269:6195–6203. https://​doi.​org/​10.​1046/j.​1432-​
1033.​2002.​03338.x
39. Kishimoto T, Tomishige N, Murate M, Ishitsuka R, Schaller
H, Mely Y, Ueda K, Kobayashi T (2020) Cholesterol asymmetry at the tip of filopodia during cell adhesion. FASEB J
34:6185–6197. https://​doi.​org/​10.​1096/​fj.​20190​0065RR

13

167

Page 14 of 15

40. Verkleij AJ, Zwaal RF, Roelofsen B, Comfurius P, Kastelijn D,
van Deenen LL (1973) The asymmetric distribution of phospholipids in the human red cell membrane. A combined study
using phospholipases and freeze-etch electron microscopy.
Biochim Biophys Acta 323:178–193. https://​doi.​org/​10.​1016/​
0005-​2736(73)​90143-0
41. Doktorova M, Symons JL, Levental I (2020) Structural andfunctional consequences of reversible lipid asymmetry in living membranes. Nat Chem Biol 16:1321–1330. https://​doi.​org/​
10.​1038/​s41589-​020-​00688-0
42. Blom TS, Koivusalo M, Kuismanen E, Kostiainen R, Somerharju P, Ikonen E (2001) Mass spectrometric analysis reveals
an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading. Biochemistry40:14635–14644. https://​doi.​org/​10.​1021/​bi015​6714
43. Samuel D, Paris S, Ailhaud G (1976) Uptake and metabolism of
fatty acids and analogues by cultured cardiac cells from chickembryo. Eur J Biochem 64:583–595. https://​doi.​org/​10.​1111/j.​
1432-​1033.​1976.​tb103​38.x
44. Rohwedder A, Zhang Q, Rudge SA, Wakelam MJ (2014)
Lipid droplet formation in response to oleic acid in Huh-7
cells is mediated by the fatty acid receptor FFAR4. J Cell Sci
127:3104–3115. https://​doi.​org/​10.​1242/​jcs.​145854
45. Targett-Adams P, Chambers D, Gledhill S, Hope RG, Coy JF,
Girod A, McLauchlan J (2003) Live cell analysis and targetingof the lipid droplet-binding adipocyte differentiation-related
protein. J Biol Chem 278:15998–16007. https://​doi.​org/​10.​1074/​
jbc.​M2112​89200
46. Wang H, Wei E, Quiroga AD, Sun X, Touret N, Lehner R (2010)
Altered lipid droplet dynamics in hepatocytes lacking triacylglycerol hydrolase expression. Mol Biol Cell 21:1991–2000.
https://​doi.​org/​10.​1091/​mbc.​e09-​05-​0364
47. Homan R, Grossman JE, Pownall HJ (1991) Differential effectsof eicosapentaenoic acid and oleic acid on lipid synthesis andsecretion by HepG2 cells. J Lipid Res 32:231–241. https://​doi.​
org/​10.​1016/​S0022-​2275(20)​42084-X
48. Schroedl NA, Hartzell CR (1984) Preferential distribution of
non-esterified fatty acids to phosphatidylcholine in the neonatal
mammalian myocardium. Biochem J 224:651–659. https://​doi.​
org/​10.​1042/​bj224​0651
49. Wang S, McLeod RS, Gordon DA, Yao Z (1996) The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases
cotranslational degradation of apolipoprotein B in transfected
COS-7 cells. J Biol Chem 271:14124–14133. https://​doi.​org/​10.​1074/​jbc.​271.​24.​14124
50. Caviglia JM, De Gomez Dumm IN, Coleman RA, Igal RA(2004) Phosphatidylcholine deficiency upregulates enzymes of
triacylglycerol metabolism in CHO cells. J Lipid Res 45:1500–
1509. https://​doi.​org/​10.​1194/​jlr.​M4000​79-​JLR200
51. Besson N, Hullin-Matsuda F, Makino A, Murate M, Lagarde
M, Pageaux JF, Kobayashi T, Delton-Vandenbroucke I (2006)
Selective incorporation of docosahexaenoic acid into lysobisphosphatidic acid in cultured THP-1 macrophages. Lipids
41:189–196. https://​doi.​org/​10.​1007/​s11745-​006-​5087-5
52. Maggio B, Ariga T, Sturtevant JM, Yu RK (1985) Thermotropic
behavior of binary mixtures of dipalmitoylphosphatidylcholine
and glycosphingolipids in aqueous dispersions. Biochim Biophys Acta 818:1–12. https://​d oi.​o rg/​1 0.​1 016/​0 005-​2 736(85)​90131-2
53. Matuoka S, Akiyama M, Yamada H, Tsuchihashi K, Gasa S(2003) Phase behavior in multilamellar vesicles of DPPC containing ganglioside GM3 with a C18:1 sphingoid base and a
24:0 acyl chain (GM3(18,24)) observed by X-ray diffraction.
Chem Phys Lipids 123:19–29. https://​doi.​org/​10.​1016/​s0009-​
3084(02)​00128-7

13

M. Murate et al.
54. Maggio B, Ariga T, Sturtevant JM, Yu RK (1985) Thermotropic behavior of glycosphingolipids in aqueous dispersions.
Biochemistry 24:1084–1092. https://​d oi.​o rg/​1 0.​1 016/​0 005-​
2736(85)​90131-2
55. Maulik PR, Shipley GG (1996) N-palmitoyl sphingomyelin
bilayers: structure and interactions with cholesterol and dipalmitoylphosphatidylcholine. Biochemistry 35:8025–8034. https://​doi.​org/​10.​1021/​bi952​8356
56. Sarmento MJ, Owen MC, Ricardo JC, Chmelova B, Davidovic
D, Mikhalyov I, Gretskaya N, Hof M, Amaro M, Vacha R, Sachl
R (2021) The impact of the glycan headgroup on the nanoscopic
segregation of gangliosides. Biophys J 120:5530–5543. https://​doi.​org/​10.​1016/j.​bpj.​2021.​11.​017
57. Palestini P, Pitto M, Sonnino S, Omodeo-Sale MF, Masserini M(1995) Spontaneous transfer of GM3 ganglioside between vesicles. Chem Phys Lipids 77:253–260. https://​doi.​org/​10.​1016/​
0009-​3084(95)​02474-w
58. Brotherus J, Renkonen O (1977) Phospholipids of subcellularorganelles isolated from cultured BHK cells. Biochim Biophys
Acta 486:243–253. https://​d oi.​o rg/​1 0.​1 016/​0 005-​2 760(77)​90020-0
59. Valiente O, Mauri L, Casellato R, Fernandez LE, Sonnino S(2001) Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM(3)by selective alkaline hydrolysis of GM3 ganglioside. J Lipid
Res 42(8):1318–1324. https://​doi.​org/​10.​1016/​S0022-​2275(20)​31583-2
60. Abe M, Makino A, Hullin-Matsuda F, Kamijo K, Ohno-Iwashita
Y, Hanada K, Mizuno H, Miyawaki A, Kobayashi T (2012) A
role for sphingomyelin-rich lipid domains in the accumulationof phosphatidylinositol 4,5-bisphosphate to the cleavage furrow
during cytokinesis. Mol Cell Biol 32:1396–1407. https://​doi.​
org/​10.​1128/​MCB.​06113-​11
61. Yamaji-Hasegawa A, Makino A, Baba T, Senoh Y, Kimura-Suda
H, Sato SB, Terada N, Ohno S, Kiyokawa E, Umeda M, Kobayashi T (2003) Oligomerization and pore formation of a sphingomyelin-specific toxin, lysenin. J Biol Chem 278(25):22762–
22770. https://​doi.​org/​10.​1074/​jbc.​M2132​09200
62. Kremer JM, Esker MW, Pathmamanoharan C, Wiersema PH(1977) Vesicles of variable diameter prepared by a modified
injection method. Biochemistry 16:3932–3935. https://​doi.​org/​
10.​1021/​bi006​36a033
63. Kobayashi T, Pagano RE (1988) ATP-dependent fusion ofliposomes with the Golgi apparatus of perforated cells. Cell
55:797–805. https://​doi.​org/​10.​1016/​0092-​8674(88)​90135-3
64. Ishitsuka R, Yamaji-Hasegawa A, Makino A, Hirabayashi Y,
Kobayashi T (2004) A lipid-specific toxin reveals heterogeneityof sphingomyelin-containing membranes. Biophys J 86:296–
307. https://​doi.​org/​10.​1016/​S0006-​3495(04)​74105-3
65. Kobayashi T, Beuchat MH, Lindsay M, Frias S, Palmiter RD,
Sakuraba H, Parton RG, Gruenberg J (1999) Late endosomal
membranes rich in lysobisphosphatidic acid regulate cholesteroltransport. Nat Cell Biol 1:113–118. https://​d oi.​o rg/​1 0.​1 038/​
10084
66. Makino A, Abe M, Murate M, Inaba T, Yilmaz N, Hullin-Matsuda F, Kishimoto T, Schieber NL, Taguchi T, Arai H, Anderluh
G, Parton RG, Kobayashi T (2015) Visualization of the heterogeneous membrane distribution of sphingomyelin associatedwith cytokinesis, cell polarity, and sphingolipidosis. FASEB J
29:477–493. https://​doi.​org/​10.​1096/​fj.​13-​247585
67. Clamme JP, Azoulay J, Mely Y (2003) Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by
two photon fluorescence correlation spectroscopy. Biophys J
84:1960–1968. https://​doi.​org/​10.​1016/​S0006-​3495(03)​75004-8
68. Azoulay J, Clamme JP, Darlix JL, Roques BP, Mely Y (2003)
Destabilization of the HIV-1 complementary sequence of
TAR by the nucleocapsid protein through activation of

Cell density‑dependent membrane distribution of ganglioside GM3 in melanoma cells﻿	

69.

70.

71.

72.

conformational fluctuations. J Mol Biol 326:691–700. https://​doi.​org/​10.​1016/​s0022-​2836(02)​01430-4
Warren SC, Margineanu A, Alibhai D, Kelly DJ, Talbot C, Alexandrov Y, Munro I, Katan M, Dunsby C, French PM (2013)
Rapid global fitting of large fluorescence lifetime imaging
microscopy datasets. PLoS ONE 8:e70687. https://​doi.​org/​10.​
1371/​journ​al.​pone.​00706​87
Becker W, Shcheslavkiy V, Frere S, Slutsky I (2014) Spatiallyresolved recording of transient fluorescence-lifetime effects
by line-scanning TCSPC. Microsc Res Tech 77(3):216–224.
https://​doi.​org/​10.​1002/​jemt.​22331
Yamaji-Hasegawa A, Murate M, Inaba T, Dohmae N, Sato
M, Fujimori F, Sako Y, Greimel P, Kobayashi T (2022) A
novel sterol-binding protein reveals heterogeneous cholesterol
distribution in neurite outgrowth and in late endosomes/lysosomes. Cellul Mol Life Sci 79:324. https://​doi.​org/​10.​1007/​
s00018-​022-​04339-6
Yokoyama K, Suzuki M, Kawashima I, Karasawa K, Nojima
S, Enomoto T, Tai T, Suzuki A, Setaka M (1997) Changes

Page 15 of 15167

in composition of newly synthesized sphingolipids of HeLa
cells during the cell cycle – suppression of sphingomyelin
and higher-glycosphingolipid synthesis and accumulation ofceramide and glucosylceramide in mitotic cells. Eur J Biochem 249:450–455. https://​doi.​org/​10.​1111/j.​1432-​1033.​1997.​
00450.x
73. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917.
https://​doi.​org/​10.​1139/​o59-​099
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