supplementary information - nature · sheng4, wonhwa cho4, koichi furukawa5, keiko furukawa6,...

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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.

Nature Chemical Biology: doi:10.1038/nchembio.2059

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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. 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. 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. 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. 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.


20


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


21

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).


22

Supplementary References

1. Chinnapen, D.J. et al. Lipid sorting by ceramide structure from plasma membrane to ER

for the cholera toxin receptor ganglioside GM1. Dev. Cell 23, 573-586 (2012).

2. Sevcsik, E. et al. GPI-anchored proteins do not reside in ordered domains in the live cell

plasma membrane. Nat. Commun. 6:6969 (2015).

3. Hewat, E. A. & Blaas, D. Structure of a neutralizing antibody bound bivalently to human

rhinovirus 2. EMBO. J. 15, 1515-1523 (1996).

4. Werner, T. C., Bunting, J. R. & Cathou, R. T. The shape of immunoglobulin G molecules

in solution. Proc. Natl. Acad. Sci. U.S.A. 69, 795-799 (1972).

5. Shibata, A. C. et al. Archipelago architecture of the focal adhesion: membrane

molecules freely enter and exit from the focal adhesion zone. Cytoskeleton 69, 380-

392 (2012).

6. Shibata, A. C. et al. Rac 1 recruitment to the archipelago structure of the focal

adhesion through the fluid membrane as revealed by single-molecule analysis.

Cytoskeleton 70, 161-177 (2013).

7. Sahl, S. J., Leutenegger, M. L., Hilbert, M., Hell, S. W. & Eggeling, C. Fast molecular

tracking maps nanoscale dynamics of plasma membrane lipids. Proc. Natl. Acad. Sci.

U.S.A. 107, 6829-6834 (2010).


supplementary information - nature · sheng4, wonhwa cho4, koichi furukawa5, keiko furukawa6,...

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