1
Supplementary Information
Raft-based interactions of gangliosides with a GPI-anchored
receptor
Naoko Komura1,2†, Kenichi G. N. Suzuki1,3†, Hiromune Ando1,2†, Miku Konishi1,2, Machi
Koikeda2, Akihiro Imamura2, Rahul Chadda1, Takahiro K. Fujiwara1, Hisae Tsuboi1, Ren
Sheng4, Wonhwa Cho4, Koichi Furukawa5, Keiko Furukawa6, Yoshio Yamauchi5, Hideharu
Ishida2, Akihiro Kusumi1,7,8*, & Makoto Kiso1,2*
1Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-
8507, Japan. 2Department of Applied Bioorganic Chemistry, Gifu University, Gifu 501-1193.
3The Institute for Stem Cell Biology and Regenerative Medicine (inStem), The National
Centre for Biological Sciences (NCBS), Bangalore, 650056, India. 4Department of Chemistry,
University of Illinois at Chicago, Chicago, 60607, USA. 5Department of Biochemistry II,
Nagoya University, Nagoya, 466-0065, Japan. 6Department of Life and Medical Sciences,
Chubu University, Nagoya, 487-8501, Japan. 7Institute for Frontier Medical Sciences, Kyoto
University, Kyoto 606-8507, Japan. 8Membrane Cooperativity Unit, Okinawa Institute of
Science and Technology, Onna-son, Okinawa 904-0412, Japan.
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Supplementary Results
Supplementary Videos
Video 1. A representative simultaneous two-color single fluorescent-molecule imaging
video clip of a 594-GM1 molecule (red) diffusing in the PM containing CD59 patches
(green) (the original video for the still image in Fig. 3a, left). The 594-GM1 molecule
diffused mostly within the CD59 patch. At the boundaries between the CD59 patch and
the bulk region, the molecule often bounced off toward inside the CD59 patch rather
than passed. It sometimes exhibited TALL within the CD59 patch. Recorded at video
rate, and replayed at a rate slowed 3-fold from real time. The scale bar is 1 m.
Video 2. The same as Video 1, except that the observed single molecule was 594-GM3 (Fig.
3a, middle).
Video 3. The same as Videos 1 and 2, except that the observed single molecule was 594-
DOPE (Fig. 3a, right). In contrast with 594-GM1 and 594-GM3 molecules shown in
Videos 1 and 2, respectively, the 594-DOPE molecule diffused mostly outside the CD59
patch, and at the boundaries between the CD59 patch and the bulk region, the
molecule often bounced off toward outside the CD59 patch rather than passed. 594-
DOPE did not exhibit TALL.
Video 4. A typical video clip of single 594-GM1 molecules diffusing in the T24-cell PM at
23˚C. The same video sequence is repeated: first without the trajectories and then
with the trajectories. Recorded at a frame rate of 2000 frames/s (a time resolution of
0.5 ms), and replayed at a rate slowed 20-fold from real time (the actual total length
of the video is 400 ms or 800 frames). The scale bar is 1 m.
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Supplementary Table 1. Kd values for binding of CTB and WGA to GM1 and
GM3, respectively.
Gangliosides† CTB/WGA Kd values
Native GM1
594-S9-GM1
594-termG6-GM1
Native GM3
594-S9-GM3
594-G6-GM3
CTB
CTB
CTB
WGA
WGA
WGA
40 ± 7 nM
44 ± 10 nM
ND*
64 ± 10 µM
67 ± 15 µM
100 ± 16 µM
*ND: not detectable
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Supplementary Table 2. The time fractions of mobile, TALL, and immobile periods,
as well as TALL durations (exponential decay lifetime), for 594-GM1 and GM3
trajectories obtained at a time resolution of 33 ms (normal video rate), inside
and outside the CD59 patches. Inside the CD59 patches, both 594-GM1 and GM3
exhibited substantial TALL fractions, which are suppressed by latrunculin-B
treatment. T24 cells were employed.
Time Fraction (%)
Location Mobile TALL* Immobile TALL (ms)‡
N†
594-GM1
Intact Cell Membrane 99.7 0.3 0 N.D.§ 162
Outside CD59 Patches 99.3 0.7 0 N.D. 202
Inside CD59 Patches 83.8 14.3 1.9 97 ± 19 246
Inside CD59 Patches
after Latrunculin B 95.8 4.2 0 N.D. 188
594-GM3
Intact Cell Membrane 99.4 0.6 0 N.D. 199
Outside CD59 Patches 99.2 0.8 0 N.D. 177
Inside CD59 Patches 86.2 12.9 0.9 124 ± 24 191
Inside CD59 Patches
after Latrunculin B 95.6 4.4 0 N.D. 189
* The detection circle radius and the threshold trapped period used were 50 nm and 80
ms (10 frames), respectively. A decrease of the detection circle radius, down to 20 nm,
reduced the TALL time fraction. Outside the CD59 patches, the TALL time fractions were
0.1% or less with a detection circle radius of 20 nm, for all molecules and all cell lines
examined here.
† The number of examined trajectories.
‡ Exponential decay lifetimes obtained by fitting the distributions of TALL periods with
single exponential functions (see Supplementary Fig. 4b). Errors indicate the fitting
errors for the 68.3% confidence limits.
§Not determined due to scarce occurrences.
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Supplementary Table 3. The time fractions of mobile, TALL, and immobile periods,
as well as TALL durations (mean value), for 594-GM1, GM3, and DOPE
trajectories obtained at a time resolution of 0.5 ms (2,000 frames/s), in the
intact PM of the PtK2, T24, NRK, and COS7 cell lines. These results show that
raft-associated GM1 and GM3 and non-raft DOPE rarely exhibit TALL in the intact
PM.
Time fraction (%)
Molecules Cell Mobile TALL* Immobile N TALL (mean)‡
(ms)
594-GM3
PtK2
98.2 1.8 0 81 5.9 ± 0.2
594-GM1 97.4 2.6 0 73 5.8 ± 0.2
594-DOPE 94.8 5.2 0 100 6.0 ± 0.2
594-GM3
T24
96.9 3.1 0 109 6.6 ± 0.2
594-GM1 97.4 2.6 0 97 6.1 ± 0.2
594-DOPE 94.9 5.1 0 98 6.5 ± 0.2
594-GM3
NRK
98.3 1.7 0 102 5.6 ± 0.1
594-GM1 97.8 2.2 0 110 5.7 ± 0.1
594-DOPE 95.4 4.6 0 97 5.9 ± 0.1
594-GM3
COS7
97.5 2.5 0 115 5.8 ± 0.1
594-GM1 97.3 2.7 0 108 5.7 ± 0.1
594-DOPE 95.0 5.0 0 110 6.0 ± 0.1
594 On glass 0 1.5 98.5 61 -
* The detection circle radius and the threshold trapped period used were 50 nm and 5 ms
(10 frames), respectively. A decrease of the detection circle radius, down to 20 nm,
reduced the TALL time fraction (0.1% or less with a detection circle radius of 20 nm for all
molecules and all cell lines examined here).
† The number of examined trajectories.
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‡ The values of the mean ± standard error, for all of the observed TALL periods. Since very
few TALL events were observed, exponential curve fitting to estimate the lifetimes was
inappropriate.
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Supplementary Table 4. The colocalization lifetimes of CD59 clusters with
ganglioside and DOPE analogues (T24 cells) and those of CD59 transient
homodimer rafts and monomers with 594-GM1 and 594-DOPE (CHO-K1 cells; no
endogenous GM1 in the PM) and 594-GM3 (GM3-depleted CHO-K1 cells).
Molecules CD59 assembly state Lifetime± S.E. (ms)† N‡
594-GM1
CD59 clusters 111 ± 8*1 239
CD59 clusters after chol. depl. 61 ± 3Y1 119
CD59 homodimer rafts 77 ± 6*2 147
CD59 monomers 49 ± 1*3,Y2 302
594-GM3
CD59 clusters 98 ± 4*4 201
CD59 clusters after chol. depl. 64 ± 4Y4 125
CD59TM clusters 42 ± 4*5,Y4 132
CD59 homodimer rafts 80 ± 6*6 157
CD59 monomers 53 ± 2*7,Y6 271
594-DOPE
CD59 clusters 57 ± 1Y1,Y4 100
CD59 homodimer rafts 38 ± 2Y2,Y6 107
CD59 monomers 40 ± 1Y3,N5,Y7 211
† Exponential decay lifetimes obtained by fitting the distributions of colocalization periods
with single exponential functions (see Fig. 4c and Fig. 5c). Errors indicate the fitting
errors for the 68.3% confidence limits.
‡ The number of examined colocalizations.
*, Y, and N. The results of the statistical test. The distribution selected as the basis for the
comparison is shown by the superscript, *. The numbers (1 - 7) indicate the different bases.
The superscript Y or N indicates that the distribution is or is not significantly different,
respectively, with t-test p values smaller or greater than 0.05, respectively.
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Supplementary Table 5. Effective diffusion coefficients on the time scale of 100
ms (D100ms) for all molecules examined in the present work in the human
epithelial T24 cell PM. The D100ms values of the ganglioside fluorescent probes were
larger than those of the non-raft phospholipids, 594-DOPE and Cy3-Fab labeled Fl-DOPE,
while a conventional GM1 marker, Cy3-labeled cholera toxin B subunit (Cy3-CTB), diffused
much more slowly than any of the other examined molecules, due to the clustering of
GM1 molecules. Single molecules of Cy3-CTB were observed within 0.5 sec after
attachment on the cell membranes, and were observed after a 10 or 20 min incubation.
This result clearly shows that CTB is not suitable for studying GM1 dynamics.
Molecules† D100ms (m2/s)
median (mean ± SE) N‡
594-S9-GM3
TMR-S9-GM3
488-S9-GM3
647N-S9-GM3
TMR-G6-GM3
594-G6-GM3
FL-S9-GM3 (Cy3-Fab labeled)
594-S9-GM1
TMR-S9-GM1
Bodipy-FL-GM1
594-termG6-GM1
Cy3-CTB within 0.5 s
after attachment to cell membrane
Cy3-CTB incubated for 10 min
Cy3-CTB incubated for 20 min
594-termG6-GD1b
594-S9-GM2
594-GN6-GM2
594-DOPE
Fl-DOPE (Cy3-Fab labeled)
0.32 (0.37 ± 0.02)*1
0.33 (0.32 ± 0.02)
0.32 (0.35 ± 0.02)
0.30 (0.34 ± 0.03)
0.32 (0.36 ± 0.03)
0.31 (0.34 ± 0.02)
0.26 (0.26 ± 0.02)
0.34 (0.36 ± 0.03)*2,N1
0.31 (0.31 ± 0.02)
0.34 (0.36 ± 0.02)
0.32 (0.34 ± 0.02)
0.20 (0.21 ± 0.03)*3,Y2
0.06 (0.11 ± 0.02)Y2Y3
0.01 (0.02 ± 0.002)Y2Y3
0.36 (0.37 ± 0.02)
0.31 (0.34 ± 0.02)
0.34 (0.35 ± 0.02)
0.20 (0.21 ± 0.02)*4,Y1,Y2
0.22 (0.23 ± 0.03)N4
103
93
145
80
94
108
100
88
107
104
231
55
56
75
228
230
169
145
81
†TMR = tetramethylrhodamine; Fl = fluorescein; CTB = cholera toxin B subunit.
*, Y, and N. The results of the statistical test. The distribution selected as the basis for the
comparison is shown by the superscript, *. Different numbers (1 - 4) indicate different bases.
The superscript Y or N indicates that the distribution is or is not significantly different,
respectively, with p values of the t-test smaller or greater than 0.01, respectively.
‡Number of examined molecules.
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Supplementary Fig. 1
Supplementary Fig. 1. WGA and CTB respectively bound to 594-S9-GM3 and GM1,
and to the native GM3 and GM1.
(a and b) Determination of the Kds for the binding of WGA and CTB to GM3 and GM1-
containing vesicles, respectively, by equilibrium SPR analysis (in 20 mM Tris-HCl, pH 7.4,
containing 0.16 M KCl). (a) CTB was injected at 5 µl/min at various concentrations (8-1,000
nM) over the sensor chip (L1) coated with either PC/GM1 (9:1) (blue) or PC/594-S9-GM1
(9:1) (red) vesicles, and the Req value at each CTB concentration was measured. The binding
isotherm was generated by plotting Req/Rmax (where Req is the response at equilibrium and
Rmax is the Req value at saturation binding) versus the protein concentration (P0). Solid lines
represent theoretical curves with Kd values determined by nonlinear least squares analysis
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of the isotherms, using the equation, Req/Rmax= 1/(1 + Kd/P0). (b) The same sequence of
measurements and analyses performed with WGA (150 µM) and vesicles made of PC/GM3
(9:1) (blue), PC/594-S9-GM3 (9:1) (red), or PC/594-G6-GM3 (9:1) (green).
Kd for the CTB binding to 594-S9-GM1 (44 ± 10 nM) was about the same as that to
GM1 (40 ± 7 nM), which suggests that the conjugation of ATTO594 to the GM1’s S9 position
does not interfere with the binding of CTB to GM1, at variance with the previous fluorescent
GM1 analogues1. Furthermore, Kd for WGA binding to 594-S9-GM3 (67 ± 15 µM) was about
the same as that to native GM3 (64 ± 10 µM), whereas that to 594-G6-GM3 (100 ± 16 µM)
was only slightly greater than that to native GM3. These results suggest that the ATTO594
conjugation to the S9 position does not interfere with the WGA binding to GM3, whereas
the conjugation to the G6 position slightly reduced the WGA affinity to GM3. It is therefore
concluded that 594-S9-GM1 and GM3 would be the optimal GM1 and GM3 analogues,
respectively.
(c) 594-S9-GM1 incorporated in the PM of live mouse B78/M2T1-1 GM1-deficient
cells formed clusters (red spots) upon the addition of fluorescein-conjugated CTB followed
by anti-fluorescein antibodies. The clusters of 594-S9-GM1 were colocalized with the green
fluorescein-CTB clusters induced by the anti-fluorescein antibodies. This result indicates that
594-S9-GM1 retains the property of CTB binding in the PM as well as in artificial lipid
membranes, as shown in (a). Similar experiments using WGA for GM3 could not be
performed because WGA binds to, in addition to GM3, various glycoproteins.
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Supplementary Fig. 2
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Supplementary Fig. 2. Cold-Triton solubility of native GM3 and GM1 as well as
exogenously incorporated fluorescent GM3 and GM1 analogues, a commercially
available GM1 probe (BodipyFL-GM1), and fluorescent phospholipid analogues.
(a) Endogenous GM1 and GM3 in T24 cells, detected in cold-Triton soluble and
insoluble fractions by high-performance thin layer chromatography. Standards: chemically
synthesized GM1 and GM3.
(b - f) Exogenously added fluorescent analogues of gangliosides and phospholipids.
Fluorescent analogues of GM3 (b), GM1 (c) with a control of fluorescently-labeled CTB, GM2
(d), GD1b (e), and phospholipids (f).
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Supplementary Fig. 3
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Supplementary Fig. 3. Colocalization of fluorescent ganglioside analogues with
the micron-scale antibody-induced CD59 patches in the live-cell PM at the level
of confocal fluorescence microscopy (representative images).
CD59 patches were induced by the addition of the anti-CD59 monoclonal antibody followed
by the secondary antibodies, in the T24-cell PMs where fluorescent GM3, GM1, or DOPE
analogues had been exogenously incorporated, and the recruitment of these analogues to
CD59 patches was monitored. The names of molecules indicated in red and blue fonts
indicate colocalized and non-colocalized molecules, respectively.
The colocalization of native GM3 or GM1 (but probably crosslinked) was examined by
adding fluorescein-conjugated CTB or an anti-GM3 monoclonal IgM antibody and
subsequently anti-mouse µ-chain specific antibodies, respectively, before the induction of
CD59 patches, in the presence of 450 mM sucrose to block the internalization of crosslinked
gangliosides (see ONLINE METHODS for details) (top rows in the groups of GM3 and
GM1). These results indicate that CD59 patches are colocalized by native GM3 and GM1,
but we could not determine whether monomeric GM3 and GM1 are also recruited to CD59
patches. Recently, when mGFP-GPI was concentrated on 3-µm-sized spots of anti-GFP
antibodies, formed by micropatterning at densities between 500 – 10,000 molecules/µm2
and occupying 0.5 – 11% of the area in the spots, CD59, another GPI-anchored protein, did
not become concentrated in the mGFP-GPI spots2. When CD59 was crosslinked by primary
and secondary antibodies as done in the present research, although the crosslinked clusters
might not be as large as 3 µm in diameter, the CD59 occupancies in the clusters were as
large as 20% or 7.7%, assuming that the average distance between CD59 molecules is 63
or 9.6 nm4 and the CD59 radius is 1.5 nm2. The high densities of CD59 employed here can
cause the concentration of the ganglioside analogues developed in this study. This is
consistent with the expected steep decrease of the diffusion coefficient of the probe
molecule when the nearest-neighbor distance of the steric obstacles was less than 10 nm
(Fig. 3d and e in Ref. 2).
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Supplementary Fig. 4
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Supplementary Fig. 4. Diffusion of 594-GM1 and GM3 outside and inside CD59
patches formed in the T24-cell PMs, observed at a time resolution of 8 ms.
(a) Typical single-molecule trajectories of 594-GM1 (top) and GM3 (bottom) outside
(left) and inside (right) CD59 patches, observed at a ~4x enhanced time resolution of 8 ms.
The trajectory length (observation duration) is shown for each trajectory. 594-GM1 and GM3
exhibited TALL (red parts of the trajectories) only within CD59 patches.
The parts of the single-molecule trajectories that were either inside or outside the
CD59 patches (only those longer than 50 consecutive frames or 400 ms were employed for
analysis) were classified into three modes of motion: (1) the all-time mobile mode, (2) the
mobile+TALL mode in which a mobile trajectory includes at least one TALL period, and (3)
the all-time immobile mode5,6. Next, the trajectories that were classified into the all-time
mobile mode and the mobile parts of the trajectories that were classfied into the mobile +
TALL mode were pooled, for analysis as the trajectories of mobile molecules
(Supplementary Table 2).
Outside the CD59 patches, virtually all of the 594-GM1 and GM3 molecules exhibited
the all-time mobile mode at an 8-ms resolution (0.5-ms-resolution data will be discussed
later). Even within CD59 patches, 594-GM1 and GM3 are quite mobile: there were no all-
time immobile trajectories (≥ 400 ms) and only ~14% (an average of GM1 and GM3) of the
overall TALL time fractions were detected (Supplementary Table 2).
(b) The duration distributions of the TALL events exhibited by 594-GM1 and GM3 in
CD59 patches were 97 and 124 ms for 594-GM1 and GM3, respectively, as the exponential
decay time constants (lifetime; the reason for the difference between 594-GM1 and GM3 is
unknown). Interestingly, partial actin depolymerization by mild latrunculin-B treatment (100
nM for 5-15 min) abolished the TALL events, suggesting that TALL within CD59 patches is
actin-dependent.
(c) The plot of the average mean-square displacement vs. the time interval for the
mobile trajectories (trajectories exhibiting the all-time mobile mode and the subtrajectories
exhibiting the mobile periods in the trajectories classified into the mobile + TALL mode) of
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594-GM1 and GM3, observed at an 8-ms resolution, inside (right) and outside (left) the
CD59 patches. All of the plots are effectively linear in the time range shown here, on the
time scale of 400 ms, indicating that the trajectories obtained during the mobile periods are
basically simple Brownian. This suggests that the effect of the fine structures that probably
exist within the CD59 patches on diffusion is averaged on this time scale, and that both the
bulk domain and CD59 patches are sufficiently large so that the bouncing effect at their
boundaries could be neglected in this time range. Note that this analysis was performed for
the CD59 patches greater than 1 x 1 µm, and that the mobile time fractions were 85% and
>99% inside and outside the CD59 patches (averages of 594-GM1 and GM3), respectively
(Supplementary Table 2).
(d) The effective diffusion coefficient determined on the time scale between 16 and
48 ms, with a midpoint at 32 ms (Deff32ms) for each individual trajectory. These results
indicate that the diffusion coefficients within CD59 patches (including TALL periods detected
at a time resolution of 8 ms) are smaller by a factor of about four than those outside CD59
patches (in the bulk domain). This reduction is not caused by the bouncing effect on 594-
GM1 and GM3 molecules at the CD59-patch boundaries: Since Deff32ms is 0.10 µm2/s inside
the CD59 patches, these molecules would cover, during 32 ms, an area of 0.013 µm2 (~110
x 110 nm), which is much smaller than the micron-sized CD59 patches (this analysis was
performed for CD59 patches larger than 1 x 1 µm), and thus the results are internally
consistent with those shown in c.
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Supplementary Fig. 5
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Supplementary Fig. 5. Partial cholesterol depletion induced much smaller CD59
patches
(a) Images of antibody-induced CD59 patches without (left) and with (right) the
preceding partial cholesterol depletion, observed by conventional fluorescence microscopy
(T24 cells). The part of the image enclosed within the box in the top image is expanded by
10-fold in the bottom image.
(b) The histograms showing the size distributions of CD59 patches without (black,
n=47) and with (red, n=63) the preceding partial cholesterol depletion. The size was
obtained after binarization of the images. This result shows that far fewer CD59 patches of
sizes greater than 0.4 µm2/s could be generated by the addition of primary and secondary
antibodies when cholesterol was depleted from the T24-cell PM before the addition of
antibodies, suggesting that the CD59 patches were not simply formed by the extensive
crosslinking of CD59 by the primary and secondary antibodies, but that smaller CD59
clusters crosslinked by antibodies were assembled by raft-lipid interactions.
(c) A model for an antibody-induced CD59 patch in micron-scales, showing that it
consists of many antibody-induced CD59 clusters (magenta circles; blue circles represent
CD59 monomers, and black and green sticks [and Y-shaped keys in the left figure] represent
primary anti CD59 monoclonal antibody and secondary antibodies, respectively), assembled
together by raft-lipid interactions. Note that the antibodies and CD59 molecules are drawn
in only two CD59 clusters (magenta circles), and abbreviated in other clusters in the figure
on the right. The figure on the right shows the top view of an antibody-induced CD59 patch
in the PM, and the figure on the left shows a close-up view of one of the antibody-induced
CD59 clusters (overhead view). Gangliosides can enter CD59 patches readily and generally
diffuse inside the patch at an average diffusion coefficient smaller by a factor of ~2
(without including the periods during TALLs) compared with that in the bulk PM (see the
trajectories as well as data in Supplementary Fig. 4). Gangliosides undergo TALLs (shown
as yellow trajectories) in an actin-dependent manner inside CD59 patches (but not outside
CD59 patches), suggesting the interaction of actin filaments with CD59 patches.
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Supplementary Fig. 6
Supplementary Fig. 6. 594-GM3 and GM1 exhibited no TALL events in the
observations at a time resolution of 0.5 ms.
Representative trajectories of 594-GM3, GM1, and DOPE incorporated in the PMs of T24 and
PtK2 cells in the resting state (without any stimulation or treatment) at 23˚C7, recorded at
a 0.5-ms resolution with a single-molecule localization precision of 17 nm. Also see
Supplementary Video 4. Only trajectories longer than 50 consecutive frames or 25 ms
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were employed for the analysis.
All-time immobile trajectories were scarcely found for all three molecules in all of the
cell types. The TALL time fractions were 1.7~3.1% for 594-GM3 and GM1 and 4.6~5.2%
for 594-DOPE in all four cell types (Supplementary Table 3). Namely, despite the probable
association of GM3 and GM1 with raft domains, they hardly exhibited TALL events just like
the non-raft 594-DOPE (or less than 594-DOPE did), at variance with the previous data by
Sahl et al.7 Based on their observations at the same time resolution of 0.5 ms (with a single-
molecule localization precision of 10~20 nm) as that employed here, Sahl and coworkers
reported that their sphingomyelin-like probe exhibited a TALL time fraction of 70%, with a
lifetime of 17 ms (12-nm apparent trapped domain diameter), whereas a phospholipid
analogue molecule showed a TALL time fraction of 30%, with a lifetime of 3 ms (22-nm
apparent trapped domain diameter), using the detection circle radius of 20~120 nm and
the threshold trapped period of 5 ms7. Here, we employed the same protocol to detect TALL
as that used by Sahl et al. with various parameters including those employed by Sahl et al.,
but we detected very minor TALL fractions (Supplementary Table 3).
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