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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2013.92
NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 11
Multiplexing DNA nanomachines to map pH changes along two intersecting
endocytic pathways inside the same cell
Souvik Modi, Clément Nizak, Sunaina Surana, Saheli Halder, Yamuna Krishnan
Supplementary Information
Supplementary Materials
Materials and Methods
All the unlabelled oligonucleotides used were obtained from Eurofins Genomics India
Pvt. Ltd. and labelled oligonucleotides (HPLC purified and lyophilized) were
obtained from IBA GmbH (Germany). Oligonucleotides were dissolved in Milli Q
water to make a 200 µM stock which was aliquoted and kept at –20oC. Depending on
the purity of fluorescently labelled oligonucleotides, they were subjected to ethanol
precipitation prior to use to remove any contaminating fluorophores.
I-switch sample preparation
5 µM of In and In′ were mixed in equimolar ratios in 20 mM potassium phosphate
buffer of desired pH containing 100 mM KCl. The resulting solution was heated to
90oC for 5 minutes, cooled to the room temperature at 5oC/15 min and equilibrated at
4oC overnight. Prior to the experiment, the solution was diluted in appropriate buffer
containing 100 mM KCl, unless mentioned.
Steady state and ratiometric measurements
Solutions of I-switch at different pH were made by diluting 1 µL of 5 µM stock
samples into 99 µL of 1× clamping buffer of desired pH. All samples were vortexed
and equilibrated for 30 min at room temperature. The experiments were performed in
a widefield microscope (Nikon Eclipse Ti-U, Nikon Japan). Cover slips containing 50
Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell
© 2013 Macmillan Publishers Limited. All rights reserved.
2
µL samples of different pH were excited at 488 nm (for A488/A647 pair) and 550 nm
(for A546/A647 pair) in a widefield microscope and emission images were acquired
using 520 nm (donor, D channel) and 669 nm (acceptor, A channel). An in vitro pH
calibration curve was obtained by plotting the ratio of donor intensity (D) at 520 nm
by acceptor intensity (A) at 669 nm (for A488/A647) and 570 nm by 670 nm (for
A546/A647 pair) as a function of pH. Mean of D/A from two independent
experiments and their SD was plotted for each pH value.
Cell culture and transfection
HeLa cells were cultured in Dulbecco’s Modified Eagle’s medium/F-12 (1:1)
(Invitrogen Corporation, USA) containing 10% heat inactivated Fetal Bovine Serum
(FBS) (Invitrogen Corporation, USA), 100 µg/mL Streptomycin and 100 U/mL
Penicillin (Invitrogen Corporation, USA). IA2.2 is a Chinese Hamster Ovary (CHO)
cell line which lacks endogenous transferrin receptors but stably expresses the human
transferrin and folate receptors. These cells were cultured in Ham’s-F12 Complete
media (HF-12, Himedia, India) containing 10% heat inactivated FBS, 100 µg/mL
Streptomycin and 100 U/mL Penicillin with 200 µg/mL G418 and 100 µg/mL
hygromycin to ensure maintenance of transferrin and folate receptors. For imaging
and transfection, cells were maintained in complete media without G418 and
hygromycin. For transient transfections, cells were plated at >50% density onto
coverslip bottomed 35 mm dish and 150 ng of ssFurin-scFv-Furin was introduced
using the Lipofectamine™ 2000 reagent system (Invitrogen Corporation, USA),
following the manufacturer’s instructions. Cells were imaged 24 h after transfection.
Immunofluorescence staining
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3
Immunofluorescence staining was carried out on scFv-Furin expressing IA2.2 cells
after fixing with 4% paraformaldehyde for 20 min at RT. To detect intracellular
antigens, the cells were permeabilized with 0.1% saponin (Sigma) in Medium 1 (M1)
buffer and stained with mouse anti-TGN46 antibody (Abcam), rabbit anti-Giantin and
rabbit anti Lamp-1 antibodies (Abcam) followed by goat anti rabbit-Cy3 conjugated
(Abcam) and goat anti mouse-Alexa 488 conjugated secondary antibodies
(Invitrogen) for 1 h, respectively.
Labelling of cells by I-switch
scFv-Furin expressing cells were washed thrice with M1 buffer prior to labelling.
Cells were incubated with endocytic tracers for indicated times in labelling medium
(complete medium). For I-switch labelling, IFuA488/A647 was diluted in labelling media
to final concentration of 500 nM while Tf conjugated switch (Tf-ITfA488/A647) was
diluted in M1 and incubated for different times at 37oC. For labelling late endosomes,
cells were incubated with IFuA488/A647 and 2 mg/mL FITC dextran in labelling media at
37oC for 1 h followed by a chase for 1 h. Sorting endosomes were labelled by 100
µg/mL Alexa-568 labelled human holo-transferrin after incubating IA2.2 cells at 37oC
for 10 min in M1 while a brief chase of 12 min marked recycling endosomes. After
incubation, excess endocytic tracers were washed off using M1 and chased for
indicated times at 37oC in complete media. TGN was labelled by incubating the cells
with IFuA546/A647 for 1.5-2 h in complete medium containing 125 µg/mL cycloheximide
followed by a chase of 1.5 h in same cycloheximide containing media.
Measurement of pH in sorting and late endosomes
scFv-Furin expressing IA2.2 cells were labelled with 500 nM IFuA546/A647 in complete
media or 500 nM Tf-ITfA488/A647 in M1 for indicated times at 37oC. Intracellular pH
© 2013 Macmillan Publishers Limited. All rights reserved.
4
gradient was abolished by addition of 50 µM nigericin and 50 µM monensin in
different pH clamping buffers ranging from pH 5 to 7.5 for 45 min. The cells were
kept in this medium until imaging, and the fluorescence ratio of donor (D, 520 nm or
570 nm as applicable) image to acceptor (A, 669 nm) image at different equilibrated
pH was calculated in individual endosomes after exciting at the donor. The mean from
the distribution of D to A ratio of individual endosome were obtained at different pH
and plotted to obtain a calibration curve. The pH of sorting endosomes, late
endosomes and TGN was estimated after labelling the respective compartments with
Tf-ITfA488/A647 and IFu
A546/A647, calculating D/A ratios and then estimating the pH value
from the calibration curve.
Image acquisition and analysis
Wide-field images were collected using Nikon eclipse Ti-U microscope (Nikon,
Japan) inverted microscope equipped with 60X, 1.4 NA objective, a metal halide
illuminator (Lumen Dynamics, Ontario, Canada), and a cooled charge-coupled device
(CCD) camera (Cascade II-512, Photometrics, Tucson, AZ, USA) controlled by
MetaMorph software (Molecular Devices, PA). Optimal dichroics, excitation, and
emission filters were used as described previously. For pH measurements, cells were
imaged in three channels to yield four images, (i) donor channel by exciting at 488 nm
and collecting at 520 nm (ii) Acceptor channel by exciting at 488 nm and collecting at
669 nm (iii) donor channel by exciting at 550 nm and collecting at 570 nm (iv)
Acceptor channel by exciting at 550 nm and collecting at 669 nm. Cross talk and
bleed-through were measured with donor only and acceptor only samples and found
to be negligible for Alexa 488-647 pair while around 25-30% of Alexa 647 was
directly excited at 550 nm excitation and subtracted from corresponding donor (A488
excitation, 520 emission image)) for representative images. Autofluorescence was
© 2013 Macmillan Publishers Limited. All rights reserved.
5
measured on unlabelled cells. All the images were then background subtracted taking
mean intensity of the cytoplasm, donor and acceptor images were co-localized and
endosomes showing co-localization were analysed using ImageJ software (NIH).
Total intensity as well as mean intensity in each endosome was measured in donor
and acceptor channels and a ratio of donor to acceptor intensities (D/A) of each
endosome was obtained. For time lapse imaging, cells were labelled as described
earlier and after incubation for 10 min with endocytic ligands, cells were imaged at 1
frame (1s exposure) per 2 seconds for a 3 to 5 minute period and compressed to 7
frames per second (fps).
© 2013 Macmillan Publishers Limited. All rights reserved.
6
Supplementary Figure S1
Steady state fluorescence measurements
Supplementary Figure S1. Representative steady state fluorescence spectra of
programmed DNA nanomachines. Fluorescence spectra of I-switch specific for (a)
Transferrin pathway and (b) Furin pathway for simultaneous pH measurements. I-
switch was diluted to 50 nM in 1X clamping buffer of pH 5.0 and 7.4, incubated for
30 min before acquiring spectra. Samples were excited at 495 nm and 545 nm
respectively and fluorescence spectra was recorded from 505 nm (for 495 nm
excitation)/555 nm (for 545 nm excitation) to 730 nm. Also shown are fluorescence
spectra of DNA nanodevices at different pH. (c) ITfA488/A647 and (d) IFu
A546/A647.
Nanodevices were diluted to 50 nM in 1X pH clamping buffer and fluorescence
Tf-ITfA488/A647
IFuA546/A647
500 550 600 650 700 750 8000
1x105
2x105
3x105
4x105
5x105
Intensity (a.u)
Waveleng th (nm)
5.0 5.25 5.5 5.75 5.95 6.27 6.5 6.75 7.0 7.25 7.4
550 600 650 700 7500
1x105
2x105
3x105
Intensity (a.u)
Waveleng th (nm)
5.0 5.25 5.55 5.75 5.95 6.27 6.5 6.75 7.0 7.4
500 550 600 650 7000
1x105
2x105
3x105
4x105
Intensity (a.u)
Waveleng th (nm)
7.4 5.0
a b H+ Salt
Salt OH-
H+ Salt
Salt OH-
H+ Salt
Salt OH-
H+ Salt
Salt OH-
550 600 650 7000
1x105
2x105
3x105
Intensity (a.u)
Waveleng th (nm)
7.4 5.0
c d
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7
spectra were acquired from 500 nm/555 nm to 730 nm by exciting at 488 nm and 546
nm respectively.
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8
Supplementary Figure S2
Characterization of scFv-Furin chimera
To check expression of scFv-Furin chimera in HeLa cells, cells were transfected with
scFv-Furin. 24 h post transfection, the cells were lysed, total protein was isolated,
resolved on SDS-PAGE and probed with anti-His tag antibody. scFv-Furin showed
two closely spaced bands near the expected size (~43-45 kDa) with negligible cross
reactivity towards both positive and negative controls (Fig. S2a). In order to study its
localization within the cell, immunofluorescence studies were carried out on cells
expressing scFv-Furin. HeLa cells expressing scFv-Furin showed a staining pattern
consistent with scFv resident in tubular compartments present in the perinuclear
region as well as in proximal small vesicles whereas untransfected controls showed
only non-specific background staining (Fig. S2b, c). When scFv-Furin expressing
cells were co-stained with anti-TGN46 antibody and anti-His Tag antibody, they
showed perfect co-localization (Fig. S2d) indicating that the expression and
localization of scFv-Furin was not disrupted due to presence of the scFv domain at the
N-terminus of Furin.
scFv-‐Furin
scFv-‐Furin
Marker
Marker
GFP-‐Furin
Untransfected
75 kDa
58 kDa50 kDa
46 kDa37 kDa
30 kDa25 kDa
scFv-‐Furin
scFv-‐Furin
Marker
Marker
GFP-‐Furin
Untransfected
75 kDa
58 kDa50 kDa
46 kDa37 kDa
30 kDa25 kDa
75 58 50
46 37
30 25
1 2 3 4 5 6 a scFv-Furin Untransfected
d
TGN46 scFv-Furin
b c
© 2013 Macmillan Publishers Limited. All rights reserved.
9
Supplementary Figure S2. Characterization of scFv-Furin chimera expression and
localization in cellulo. (a) Western blot analysis of scFv-Furin post transfection in
HeLa cells probed using anti His-Tag antibody. Lane 1: scFv-Furin, 2: Marker, 3:
scFv Furin (lower concentration), 4: Marker 5: GFP transfected HeLa cells 6:
Untransfected HeLa cells. (b) scFv-Furin transfected and (c) untransfected HeLa cells
were fixed and stained with mouse anti His-tag and Myc-tag antibodies and then
probed with fluorescein conjugated goat anti mouse secondary antibody. (d) scFv-
Furin transfected HeLa cells treated with mouse anti His-tag and rabbit anti TGN46
antibody to mark scFv-Furin and TGN46 respectively and then probed with the
relevant secondary antibodies. Individual cells have been demarcated by a white
outline. Scale bar: 10 µm.
© 2013 Macmillan Publishers Limited. All rights reserved.
10
Supplementary Figure S3
scFv of the scFv-Furin functions as an artificial receptor for I-switch
In order to confirm whether the scFv domain is capable of recognizing and
endocytosing I-switch from the extracellular milieu, scFv-Furin expressing HeLa cells
were incubated with 500 nM ITfA488/A647 in the external medium for ~1 h, washed and
chased. It was seen that ITfA488/A647 was distributed in numerous small punctate
vesicles throughout the cytoplasm that gradually concentrated into a tubular
compartment occupying the perinuclear region. This uptake was absent in
untransfected or mock transfected cells (Fig. S3a, c). These findings were
recapitulated when the same experiment was performed on TRVb-1 cells transfected
with scFv-Furin indicating that this uptake by scFv-Furin is not cell type specific
(Fig. S3b, d). Cumulatively, this demonstrates that the uptake of ITfA488/A647 is
dependent only on the presence of the scFv-Furin. In order to check whether uptake
by the scFv-Furin is specific to the DNA assembly (i.e., the I-switch), a competition
assay was carried out. Endocytic uptake in HeLa cells expressing scFv-Furin was
measured as described for ITfA488/A647 (500 nM) alone and in the presence of 50 fold
excess (25 µM) of a random, non competitive sequence. It was seen that uptake of
ITfA488/A647 remained unaffected (Fig. S3e, f). However when the same experiment was
carried out in the presence of 50 fold excess of Icomp, uptake was dramatically
decreased indicating that Icomp had sequence specifically competed out ITfA488/A647.
This confirmed scFv-Furin acts as a receptor for I-switch containing the d(AT)4 tag.
© 2013 Macmillan Publishers Limited. All rights reserved.
11
Supplementary Figure S3. scFv domain of scFv-Furin functions as an artificial
receptor for I-switch. scFv-Furin expressing (a) HeLa cells and (b) TRVb-1 cells
labelled with 500 nM ITfA488/A647 for 1 h at 37°C, washed, chased for 3 h and then
imaged. (c, d) Quantification of images represented in (a-b). Mean intensity at 520
nm of 20 cells ± SEM is shown. Af = Autofluorescence, Un = Untransfected, scFv =
scFv-Furin expressing cells. Individual cells have been demarcated by a white outline.
(e) Schematic of competitive uptake of I-switch by scFv-Furin expressing HeLa cells.
ITfA488/A647 uptake (1): in the absence of competitor, (2): in the presence of 25 µM of a
dsDNA sequence that lacks the d(AT)4 tag, (3): in the presence of 25 µM of ITf
dsDNA. (f) Mean fluorescence intensity at 647 nm normalized with respect to (1) and
presented as percentage intensity of internalized ITfA488/A647. All the experiments were
performed in triplicate. Scale bar: 10 µm.
Hela
TRVb-1
Autofluores c enc e C ontro l s c F v-‐F urin0
200
400
600
800
1000
1200
1400
Mea
n In
tensity (a.u)
Autofluores c enc e c ontro l s c F v-‐F urin0
200
400
600
800
1000
1200
1400
Mea
n in
tensity (a.u)
C ontrol R andom S equenc e Unlabaled S equenc e0
20
40
60
80
100
120
Per
centage Intensity
1 2 3
1 2 3
a
Untransfectedddd scFV-Furin b
scFV-Furin Af Un scFv
d
f
Untransfected
c
e Af Un scFv
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12
Supplementary Figure S4
I-switch traffics onwards from sorting and late endosomes
After endocytosis, Furin traffics from early/sorting endosome to late endosome and
finally accumulates in the trans-Golgi network. To check whether IFu is still resident
in these organelles, scFv-Furin expressing HeLa cells were labelled with a mixture of
Tfn568 and IFuA488/A647 for 10 min and chased for 2 h. Co-localization between the two
markers was lost, additionally confirmed by Pearson’s correlation coefficient,
showing that at this time point IFu had trafficked forward from early/sorting
endosomes (Fig. S4a, d). Trafficking of IFu onwards from late endosomes was
analyzed by co-pulsing cells with FITC-Dextran and IFuA546/A647 for 2 h in presence of
cycloheximide followed by a chase of 1 h in cycloheximide containing media. In
control cells similar labelling protocol was used in absence of cycloheximide to
confirm late endosomal accumulation. Pearson’s coefficient revealed that in absence
of cycloheximide, IFu is localized in late endosomes whereas in presence of
cycloheximide, IFu is transported out from late endosomes (Fig. S4b, c, e).
Tfn568
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13
Tfn568
IFu
I555F-Dex IFu-CHX
F-Dex IFu+CHX
a
b
c
SupplementaryFigure S4. Trafficking of I-switch onwards from sorting and late
endosomes. (a) scFv-Furin expressing HeLa cells pulsed with IFuA488/A647 and
Transferrin-A568 (Tfn568) for 10 min at 37oC, washed, chased for 2 h and imaged. (b,
c) scFv-Furin expressing IA2.2 cells co-pulsed with IFuA546/A647 and FITC-Dextran (F-
Dex) in absence of cycloheximide (b) and in presence of 100 µg/mL cycloheximide
(c) and chased for 1h in absence (-CHX) or presence of cycloheximide (+CHX),
washed and imaged in a widefield microscope. (d, e) Quantification of co-localization
between IFu and endosomal markers used in a and b. Arrowheads indicate regions
shown in insets. Individual cells have been demarcated by a white outline.
Experiments were performed in duplicate. Error bar: Mean±SD. Scale Bar: 5µm.
-‐C HX +C HX-‐0.4-‐0.20.00.20.40.60.8
Pea
rson's C
oefficien
t
a
Tfn568 scFv-‐Furin -‐0.2
0.0
0.2
0.4
0.6
0.8
Pears
on's coefficient
d Pixel shift
Coloc
F-‐Dex scFv-‐Furin +CHX
c
e
scFv-‐Furin F-‐Dex -CHX b
© 2013 Macmillan Publishers Limited. All rights reserved.
14
Supplementary Figure S5
scFv-Furin does not localize in lysosomes or the cis-Golgi
In order to confirm the identity of compartments containing IFu that had trafficked
onwards from late endosomes, co-localization was carried out with (a) lysosomal
(Lamp-1) and (b) cis-Golgi (Giantin) markers. To confirm scFv-Furin localization,
scFv-Furin expressing IA2.2 cells were labelled with IFuA546/A647 as described earlier
and fixed using 4% PFA and stained using anti Lamp-1 and anti Giantin antibodies.
It was observed that IFu did not co-localize with Lamp-1 (Fig. S5a) or Giantin (Fig.
S5b), indicating that IFu at this stage is not in the cis-Golgi or the lysosome. To
confirm its localization in TGN, we labelled cells with NBD-C6-Ceramide for 30 min
in M1 buffer in presence of 125µg/mL cycloheximide and chased for 30 min in
cycloheximide containing M1 buffer NBD-C6-Ceramide showed co-localization with
IFu. This confirms that in presence of cycloheximide, scFv-Furin traffics out of late
endosomes into the TGN (Fig. S5c).
© 2013 Macmillan Publishers Limited. All rights reserved.
15
Supplementary Figure S5. Retrograde transport of I-switch into the trans-Golgi
network. scFv-Furin expressing IA2.2 cells pulsed with IFuA488/A647 for 2 h in presence
of 125 µg/mL cycloheximide and chased for 1.5 h in presence of cycloheximide,
washed and fixed using 4% PFA in M1 buffer. Cells were then probed with (a) Rabbit
anti Lamp-1 antibody, (b) Rabbit anti Giantin antibody followed by Cy3 conjugated
secondary antibodies and imaged in a confocal microscope. (c) TGN localization of
IFu at indicated time. Cells were pulsed and chased with IFuA546/A647 as described
earlier and stained with NBD-C6-ceramide for 30 min, chased for 30 min in
cycloheximide containing M1 buffer at 37oC and imaged in a confocal microscope.
NBD-C6-Ceramide
+CHX c
I555
Giantin
b
I647
I647 LAMP-1
+CHX a
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16
Individual cells have been demarcated by a white outline. Scale bar: (a) 5 µm (b) 2
µm and (c) 10 µm.
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17
Supplementary Figure S6
Stability of scFv-Furin inside late endosomes
The C-terminus of Furin has a signal sequence that functions to localize Furin to the
TGN. The N-terminus of Furin bears the scFv domain which binds the DNA pH
reporter (Fig. S6a). Thus the functional readout of an intact C-terminus is the ability
of scFv-Furin to localize in the TGN. The functional readout of an intact N-terminus
is the transport of the pH sensor to a given organelle and the resultant altered pH
readout.
Consider IA2.2 cells whose TGN has been labelled as described in the main
manuscript (Fig. 2c, h) to be in state B. Cells in State B can be induced to achieve a
State A, by just washing out cycloheximide and incubating the cells in culture
medium devoid of cycloheximide (Fig. S6b). Here, the scFv-Furin chimera simply
cycles out of the TGN and accumulates in late endosomes (Fig. S6c-e). No co-
localization was observed with other endocytic markers such as anti-Lamp-1, anti-
TGN46 or transferrin (data not shown). This reveals that the N-terminal domain
comprising scFv is intact, as the I-switch is transported to the LEs along with scFv-
Furin. This is reaffirmed by the altered pH readout of 5.5.
Cells can be returned to State B again, by simply incubating cells in State A in
cycloheximide for 1 h. This is confirmed by immunofluorescence with anti-TGN 46
(Fig. S6f-h). No co-localization was observed with other endocytic markers such as
anti-Lamp-1, FITC-Dextran or transferrin (data not shown). This reveals that after
more than 3 h, the C-terminus of scFv-Furin containing the localization signal
required for transport to the TGN is intact. It also reveals that the N-terminal scFv
domain is also intact, as indicated by the transport of the I-switch to the TGN and
change in the corresponding pH readout to pH 6.1.
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18
All the studies reported in the manuscript were performed within 1 h of pulsing the
cells with I-switch. Multiple cyclings were never used and are presented here merely
as an indication of scFv-Furin intactness and functionality.
!
!
!
!
!
!
!
!
!
!
!
scFv-Furin chimera
a
b
!
No CHX or remove CHX 45 mins to 1 hour
Add CHX 1 hour
0.000.250.500.75
Pear
son'
s co
effic
ient
Coloc Pixel shift
c
f
0.000.250.500.75
Pear
son'
s co
effic
ient
Coloc Pixel shift
e
h
d
g
IFu F-Dex
IFu TGN46
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19
Supplementary Figure S6. Stability of scFv-Furin chimera inside late endosomes.
(a) Schematic of stability assay of scFv-Furin chimera complexed with IFu inside cells.
(b) Schematic of recycling assay between late endosome and TGN. Post labelling of
late endosomes (State A) in IA2.2 cells with I-switch (red rod), incubation in
cycloheximide (CHX) for 1 h, results in localization of the I-switch-scFv-Furin
complex in the TGN (State B). Washing out cycloheximide from cells in State B
followed by incubation in culture medium devoid of cycloheximide results in the
localization of the I-switch-scFv-Furin complex in the late endosomes (State A).
Cells may be cycled between these two states at least twice, showing quantitative re-
positioning of I-switch from LE to TGN, TGN to LE and back again. (c-e) State A:
Late endosomal localization of the I-switch after washing out cycloheximide from
IA2.2 cells in state A. (c) I-switch co-localizes with FITC-Dextran (LE marker) (d)
Associated Pearson’s correlation co-efficient (e) Corresponding pH read-out (pH 5.5)
confirming the intactness of N-terminus of scFv-Furin chimera. (f-h) State B: TGN
localization of the I-switch upon incubation of cells in State B in cycloheximide for
45 min. (f) I-switch co-localized with anti-TGN46 (g) Associated Pearson’s
correlation co-efficient (h) Corresponding pH read-out (pH 6.1) confirming the
intactness of C-terminus of scFv-Furin chimera (localization to TGN) and N-terminus
(I-switch transport to TGN also revealed in altered pH readout). Individual cells have
been demarcated by a white outline. Scale bar: 10 µm.
© 2013 Macmillan Publishers Limited. All rights reserved.
20
Supplementary Figure S7
Conjugation of ITf with Transferrin
ITf was conjugated with human holo-transferrin using a hetero bi-functional
crosslinker (Sulfosuccinimidyl-6-(3'-[2-pyridyldithio]-propionamido) hexanoate) or
sulfo-LC-SPDP. Briefly, transferrin was conjugated to sulfo-LC-SPDP in PBS-EDTA
(20 mM Na-Phosphate buffer pH 7.4, 1 mM EDTA) at room temperature for 6 h.
Conjugated transferrin-SPDP (Tf-SPDP) was purified using a 30 kDa Amicon.
Amount of SPDP conjugation was quantified and a 2-5 mole SPDP/Tf was obtained
which was further conjugated to thiol modified ITf by mixing them in a 1:2.5 to 1:5
ratio in PBS-EDTA followed by 24 h incubation at 4oC. Formation of Tf-ITf conjugate
was assayed using 3% Agarose-TAE gel.
Supplementary Figure S7. Conjugation of ITfA488/A647 with transferrin. Thiolated I-
switch was conjugated with SPDP-modified transferrin at 4˚C overnight. Different
species were then resolved in a 3% Agarose-TAE gel run at RT. Lanes: 1: ITfA488/A647,
2: 1:4 ITfA488/A647:Tf-SPDP, 3: 1:2.5 ITf
A488/A647:Tf-SPDP.
ssDNA ITf
A488/A647-SH
(ITfA488/A647-S)2
Tf-ITfA488/A647
1 2 3
dsDNA
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21
Supplementary Figures S8 and S9
Purification of Tf-SPDP conjugate
Supplementary Figure S8. Size exclusion chromatography (SEC) purification of
DNA conjugates. I-switch conjugates were injected in a SEC-HPLC and separated
using an isocratic flow of PBS, pH 7.4 over 16 min. Samples were monitored by their
absorbance at 260 nm. Vo (void volume) and exclusion limit Vex were measured by
injecting Blue-Dextran and ATP respectively. 1 and 2: Fractions collected and
analyzed further using gel electrophoresis.
0
2
4
6
0
4
8
12
4 8 12 160
3
6
9
T ime (min)
IT fA488/A647
Intensity (a.u) x 10
4
T f
T f-‐IT fA488/A647
V0 Vex
Py 2-Thione
ITfA488/A647
Tf
Tf-ITfA488/A647
1 2
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22
Supplementary Figure S9. Characterization of Tf-ITf conjugate separated by SEC-
HPLC. SEC fractions collected at indicated times were resolved in 3% Agarose-TAE
run at RT. Lanes: 1.Thiolated I-switch, 2. SEC fraction eluted at retention time Rt 8
min.
1
Tf-ITfA488/A647
(ITfA488/A647-S)2
ITfA488/A647-SH
2
© 2013 Macmillan Publishers Limited. All rights reserved.
23
Supplementary Figure S10
Strategies of “SimpHony”
Supplementary Figure S10. Schematic of pulse and chase involved for ‘SimpHony’.
(a) Sequential pulse. Late endosomes are labelled first with 500 nM IFuA546/A647 for 30
min in complete media at 37oC and chased for 45 min. To mark early/sorting and
recycling endosomes of same cells, a second pulse of 500 nM Tf-ITfA488/A647 in M1
buffer was introduced for 10 min at 37oC. (b) Simultaneous pulse. scFv-Furin
expressing IA2.2 cells are labelled for 10 min with a mixture of Tf-ITfA488/A647 and
IFuA546/A647 (500 nM each) in M1 buffer at 37oC.
Co-pulse, 10 min
Tf-ITfA488/A647 + IFu
A546/A647
Simultaneous pulse
B
Sequential pulse
30 min
Pulse
45 min,
Opti-MEM/M1
Chase
LE
IFuA546/A647
A
EE
10 min, M1Pulse
Tf-ITfA488/A647
EE
LE
Chase LERE
12 min, Comp media
Pulse 1
Pulse 2
EE
a b
© 2013 Macmillan Publishers Limited. All rights reserved.
24
Supplementary Figure S11
Simultaneous pH mapping of RE and LE
Simultaneous marking of recycling endosomes and late endosomes was achieved by
labelling scFv-Furin expressing IA2.2 cells with 500 nM IFuA546/A647
in complete
media for 30 min and chasing for 45 min. Recycling endosomes in the same set of
cells were marked by pulsing 500 nM Tf-ITfA488/A647
for 10 min followed by a 12 min
chase in M1 buffer.
Supplementary Figure S11. “SimpHony” of recycling endosomes and late
endosomes. scFv-Furin expressing IA2.2 cells were pulsed with IFuA546/A647 for 30
min, chased for 45 min followed by a second pulse of Tf-ITfA488/A647 for 10 min and a
12 min chase, washed and imaged in a widefield microscope. Tf-ITf positive
endosomes are shown in magenta and blue while IFu positive endosomes are
represented in red and green respectively. Individual cells have been shown using a
white outline. Scale bar: 5 µm.
RE
LE
A546
A488 FRET647
FRET647
SimpHony
© 2013 Macmillan Publishers Limited. All rights reserved.
25
TGN
EE
SimpHonyA488 FRET647
A546 FRET647
Supplementary Figure S12
Simultaneous pH mapping of EE and TGN
Simultaneous marking of early/sorting endosomes and TGN was achieved by two step
labelling. scFv-Furin expressing IA2.2 cells were pulsed with 500 nM IFuA546/A647
in
complete media containing 125 µg/mL cycloheximide for 2 h, chased in the same
medium for 90 min to achieve TGN accumulation. Sorting endosomes in the same set
of cells were marked by pulsing 500 nM Tf-ITfA488/A647
for 10 min in M1 buffer
containing 125 µg/mL cycloheximide.
Supplementary Figure S12. “SimpHony” of early/sorting endosomes and TGN.
scFv-Furin expressing IA2.2 cells were pulsed with IFuA546/A647 for 120 min in
presence of 125 µg/mL cycloheximide, chased for 90 min in presence of
cycloheximide, followed by a second pulse of Tf-ITfA488/A647 for 10 min, washed and
imaged in a widefield microscope. Tf-ITf positive endosomes are shown in magenta
and blue while IFu positive TGN is represented in red and green respectively.
Individual cells have been demarcated using a white outline. Scale bar: 5 µm.
© 2013 Macmillan Publishers Limited. All rights reserved.
26
Supplementary Figure S13
Simultaneous pH mapping of RE and TGN
Simultaneous marking of recycling endosomes and TGN was achieved by a two step
labelling. scFv-Furin expressing IA2.2 cells were pulsed with 500 nM IFuA546/A647
in
complete media containing 125 µg/mL cycloheximide for 2h, chased in the same
medium for 90 min to achieve TGN accumulation. Recycling endosomes in the same
set of cells were marked by pulsing 500 nM Tf-ITfA488/A647
for 10 min in M1 buffer
containing 125 µg/mL cycloheximide and chased for 12 min in the same buffer.
Supplementary Figure S13. “SimpHony” of recycling endosomes and TGN. scFv-
Furin expressing IA2.2 cells were pulsed with IFuA546/A647 for 120 min in presence of
125 µg/mL cycloheximide, chased for 90 min in presence of cycloheximide,
followed by a second pulse of Tf-ITfA488/A647 for 10 min and a 12 min chase, washed
and imaged in a widefield microscope. Tf-ITf positive endosomes are shown in
magenta and blue while IFu positive TGN are represented in red and green
respectively. The white outline demarcates the single cell of interest. Scale bar: 5 µm.
RE
TGN
A488
A546
FRET647
FRET647
SimpHony
© 2013 Macmillan Publishers Limited. All rights reserved.
27
Supplementary Figure S14
Dynasore blocks early endosomal fission
Supplementary Figure S14. Dynasore mediated arrest of endosomal fission. (a)
Early/sorting and late endosomes in IA2.2 cells expressing scFv-Furin were marked
with Tf-ITfA488/A647 at 37oC for 10 min. Time lapse images of control cells in (-Dy)
Absence of dynasore (+D) In presence of 160 µM dynasore and (W) After dynasore
was washed out and incubated for 10 min at 37oC for recovery. Images were acquired
at a time interval of 2s over a 2 min period and compressed to a movie played at 7 fps.
Images were acquired with 1 s exposure with identical microscope and camera settings
and represented with the same image brightness and contrast functions. (b) An ROI
where tubular endosomes are predominant is shown for better representation.
a
b
+Dy
W
-Dy
© 2013 Macmillan Publishers Limited. All rights reserved.
28
IFuTfn568
a
Tfn568 EGFP-Furin
b
Supplementary Figure S15
Effect of brefeldin A on early endosome and TGN
Supplementary Figure S15. Furin containing tubules do not co-localize with
transferrin. (a) scFv-Furin expressing cells were pulsed with IFuA488/647 for 2 h and
chased for 30 min in presence of cycloheximide to label TGN, followed by brefeldin
A treatment for 10 min at 37oC. (b) EGFP-Furin expressing cells were treated with
brefeldin A for 10 min at. Cells were pulsed with Tfn568 in presence of brefeldin A
and cycloheximide to label early endosomes. Scale bar: 10 µm.
© 2013 Macmillan Publishers Limited. All rights reserved.
29
Supplementary Figure S16
Localization of DNA nanoswitches in compartments after brefeldin A treatment
Supplementary Figure S16. Localization of Tf-ITf and IFu post brefeldin A treatment.
scFv-Furin expressing IA2.2 cells were pulsed with IFuA546/647 for 2 h and chased for
30 min in presence of cycloheximide followed by absence (a) or presence (b) of BFA
(20 µg/mL) treatment for 10 min at 37oC. Cells were pulsed with Tf-ITfA488/A647 in
presence of brefeldin A and cycloheximide to label early endosomes. Arrowheads
show microtubule organizing centre. The white outline shows the cell of interest.
Scale Bar: 10 µm
a
b
Tf-ITfA488
Tf-ITfA488
IFuA546
IFuA546
-BFA
+BFA
© 2013 Macmillan Publishers Limited. All rights reserved.
30
IFu
IFu
TGN46
TGN46
-BFA
+BFA
*
a
b
Supplementary Figure S17
DNA nanoswitches redistribute in tubules positive for TGN46 after brefeldin A
treatment
Supplementary Figure S17. ScFv-Furin expressing IA2.2 cells were pulsed with
IFuA488/647 for 2 h and chased for 30 min in presence of cycloheximide followed by
absence or (a) presence (b) of brefeldin A (20 µg/mL) treatment of 10 min at 37oC.
Cells were fixed and stained with mouse TGN46 primary antibody followed by
Texas-Red conjugated secondary antibodies. Cell of interest is demarcated using a
white outline. Scale Bar: 10 µm.
© 2013 Macmillan Publishers Limited. All rights reserved.
31
Supplementary Figure S18
D/A heat maps of Tf-ITfA488/A647 pre and post treatment with brefeldin A
Supplementary Figure S18. D/A heat map of cells labelled by Tf-ITfA488/A647 post
labelling with IFuA546/A647 followed by brefeldin A treatment for 10 min at 37oC.
pH
5.0
7.0
© 2013 Macmillan Publishers Limited. All rights reserved.
32
Supplementary Figure S19
Three organelle SimpHony captures differential pH heterogeneity
Apart from Tf-ITfA488/A647 which labels early endosomes in brefeldin A treated cells,
we used a second internal control in the same cells where treatment-induced tubule
formations are predominant. Labelling of the TGN with IFuA546/A647 shows ~85-90%
IFuA546/A647 is resident in TGN while 10-15% IFu
A546/A647 is present in small punctate
vesicles that could be endosomes that have not yet trafficked forward to the TGN.
These endosomes showed a pH of 5.89±0.16 in the absence of brefeldin A. In the
presence of brefeldin A, the pH of this vesicular population was pH 6.02±0.09 and
does not change significantly (Fig. S19e, f). However, pH of the tubular regions
contain IFu and TGN markers change significantly to 6.35±0.17 from 5.94 ± 0.15 (Fig.
S19c, d).
Supplementary Figure S19. pH heterogeneity of early endosomes and TGN pre and
post treatment with brefeldin A. IA2.2 cells were pulsed with IFuA546/A647 for 2 h and
chased for 30 min in presence of cycloheximide followed by absence or presence of
0
50
100
150
200 -‐B F A
Fre
quen
cy
A
5.0 6.0 6.5
0
5
10
15
20
25
30
35 -‐B F A
Fre
quen
cy
D0
5
10
15
20
25
30 -‐B F A
Fre
quen
cy
ves ic les
0
50
100
150
200
250
5.0 6.0 6.55.0 6.0 6.5
+B F A
Fre
quen
cy
pH
0
5
10
15
20 +B F A
Fre
quen
cy
pH
0
5
10
15
20
25
30 +B F A
Fre
quen
cy
pH
a
Tf-ITfA488/A647
EE
b
Tf-ITfA488/A647
EE
c
IFuA546/A647
TGN
d
IFuA546/A647
Tubule
e
IFuA546/A647
Vesicles
f
IFuA546/A647
Vesicles
© 2013 Macmillan Publishers Limited. All rights reserved.
33
brefeldin (20 µg/mL) for 10 min at 37oC. Cells were further treated with Tf-ITfA488/A647
in presence of brefeldin A and cycloheximide to label early endosomes.
© 2013 Macmillan Publishers Limited. All rights reserved.
34
Supplementary Figures S20 and S21
Effects of noise and signal/noise ratio on pH estimates
In order to discount the contribution of noise and signal/noise ratio (S/N) to pH
estimates, especially in areas of low fluorescence intensity, endosomes of diverse
intensity were chosen, without any bias to endosomes of specific intensities. Fig. S20a
shows a typical scatter plot of mean donor intensity (D) with observed mean acceptor
intensity (A) at both extreme pH values (pH 5.0 and 7.2) (Fig. S20a, c). The
symmetrical distribution of intensities about the slope without particular scatter either
towards high D or low A is noteworthy. In fact one can fit the scatter with a straight
line passing through the origin and the slope obtained from the plot are in good
agreement with the mean D/A obtained from the total endosomes.
S/N at the transition pH of a given I-switch is equally important and to rule out any
contribution of pH estimates, two typical scatter plots at pH1/2 (Fig. S20 b, d) were
plotted. Here we have taken mean donor intensity (D) and plotted it as a function of
D/A, to demonstrate that even if the data was analyzed using a method different from
that shown in Fig. S20a and c, S/N is a non-issue for these systems. A symmetrical
distribution over mean D/A at lower donor intensity confirmed that even at transition
pH, DNA nanodevices show minimal effect of low donor intensity. The Y-axis
regimes are chosen to span the complete range of D/A that can be exhibited by the
respective I-switch.
To check the number of endosomes that show altered D/A due to their low donor or
acceptor intensities, we correlated these donor and acceptor intensities with individual
endosomal D/A in a 2D scatter plot.
We clamped the pH of endosomes labelled with FITC-Dextran and TMR Dextran to
pH 6.0 and analyzed ~180 endosomes by the above method (this is done by taking the
© 2013 Macmillan Publishers Limited. All rights reserved.
35
0 5000 10000 15000 200000
3
6
9 pH 6.0
D/A
D
0 4000 8000 120000
5
10
15
20
25
30 pH 6.25
D/A
D
0 5000 10000 15000 20000 250000
3000
6000
9000
12000 pH 5.0 pH 7.2
Acc
epto
r
Donor
IFuA546/A647
0 5000 10000 15000 200000
1000
2000
3000
4000 pH 7.2 pH 5.0
Acc
epto
r
Donor
Tf-ITfA488/A647
a b
c d
ratio of FITC and TMR and plotting FITC & TMR intensities versus the FITC/TMR
ratio). The FITC/TMR ratio shows a Gaussian spread as expected, with ~3% of
endosomes falling outside the range of the mean ± 2 SD (SD = standard deviation)
(data not shown). We use this parameter of mean ± 2 SD as a threshold for the
analysis of pH clamped endosomes labelled with Tf-ITf and IFu to estimate the
proportion of outliers, and if this is significant, to what extent it contributes to the
errors/spread in the pH measurement.
IFu Tf-ITf
pH 5 ~1.7% (4 out of 225) pH 5.0 ~1.6% (2 out of 124)
pH 5.5 ~4.6 % (9 out of 193) pH 6.25 ~ 4.7% (7 out of 143)
pH 6 ~ 3.6% (7 out of 194) pH 6.5 ~4% (5 out of 124)
All experimental pH measurements are on organelles that show pH < 6.5 under any
condition. It is apparent that less than 10% of endosomes fall outside the significance
range. Hence due to their small numbers, their contribution to the spread of the mean
pH population is negligible.
© 2013 Macmillan Publishers Limited. All rights reserved.
36
Supplementary Figure S20. Effect of signal/noise ratio on D/A at different pH
values. (a) Scatter plot of donor (D) versus acceptor (A) intensities for Tf-ITfA488/A647.
(b) Scatter plot of D/A with D at the transition pH of Tf-ITfA488/A647. (c) Scatter plot of
D versus A for IFuA546/A647. (d) Analogous scatter plot of D/A with D at the transition
pH of IFuA546/A647.
Supplementary Figure S21. Effect of donor and acceptor intensities on D/A ratio. A
2D D/A scatter with respect to mean donor and acceptor intensities was plotted. Mean
donor is represented in red and mean acceptor is represented in blue. Mean ± 2SD
with respect to mean is represented as solid lines. Any endosomes that fall outside this
threshold are considered as outliers.
FITC
0 1 2 3 40
2000
4000
6000
8000
10000
12000
14000
16000
18000
F IT C TMR
F /R
FITC
0
5000
10000
15000
20000
25000
TMR
pH 6
0 1 2 3 4 5 6 70
5000
10000
15000
20000
25000
D onor A cceptor
D/A
Donor
0
5000
10000
15000
20000
25000
30000
35000
Acc
epto
r
0 1 2 3 4 5 6 70
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
D onor A cceptor
D/A
Donor
0
5000
10000
15000
20000
Acc
epto
r
0 1 2 3 4 5 6 70
10000
20000
D onor A cceptor
D/A
Donor
0
2000
4000
6000
8000
10000
12000
14000
16000
Acc
epto
r
pH 5.0
pH 5.5
pH 6.0
0 5 10 15 20 250
500
1000
1500
2000
2500
3000
3500
4000
D onor A cceptor
D/ADonor
0
700
1400
Acc
epto
r
pH 5.0
0 5 10 15 20 250
2000
4000
6000
8000
10000
12000
D onor A cceptor
D/A
Donor
0
200
400
600
800
1000
1200
1400
Acc
epto
r
0 5 10 15 20 250
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
D onor A cceptor
D/A
Donor
0
200
400
600
800
1000
1200
1400
1600
Acc
epto
r
pH 6.5
pH 6.25
IFuA546/647 Tf-ITf
A488/647
FITC
0 1 2 3 40
2000
4000
6000
8000
10000
12000
14000
16000
18000
F IT C TMR
F /R
FITC
0
5000
10000
15000
20000
25000
TMR
pH 6
0 1 2 3 4 5 6 70
5000
10000
15000
20000
25000
D onor A cceptor
D/A
Donor
0
5000
10000
15000
20000
25000
30000
35000
Acc
epto
r
0 1 2 3 4 5 6 70
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
D onor A cceptor
D/A
Donor
0
5000
10000
15000
20000
Acc
epto
r
0 1 2 3 4 5 6 70
10000
20000
D onor A cceptor
D/A
Donor
0
2000
4000
6000
8000
10000
12000
14000
16000
Acc
epto
r
pH 5.0
pH 5.5
pH 6.0
0 5 10 15 20 250
500
1000
1500
2000
2500
3000
3500
4000
D onor A cceptor
D/ADonor
0
700
1400
Acc
epto
r
pH 5.0
0 5 10 15 20 250
2000
4000
6000
8000
10000
12000
D onor A cceptor
D/A
Donor
0
200
400
600
800
1000
1200
1400
Acc
epto
r
0 5 10 15 20 250
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
D onor A cceptor
D/A
Donor
0
200
400
600
800
1000
1200
1400
1600
Acc
epto
r
pH 6.5
pH 6.25
IFuA546/647 Tf-ITf
A488/647
© 2013 Macmillan Publishers Limited. All rights reserved.
37
Supplementary Table S1. Sequences of oligonucleotides used
Name Sequence
IFuA488 5’-Alexa488—CCCCTAACCCCTAACCCCTAACCCCATATATATCCTAGAACGACAGACAAACAGTGAGTC-3’
IFu′A647
5’-GACTCACTGTTTGTCTGTCGTTCTAGGATATATATTTTGTTATGTGTTATGTGTTAT-3’
Tf-con 1 5’- Alexa 488--CCCCTAACCCCTAACCCCTAACCCCTTTAAATAGGCACCGGCATGCGCAGTCTGACGT
Tf-con 2′
5’—Thiol--ACGTCAGACTGCGCATGCCGGTGCCTATTTAAATTTGTTATGTGTTATGTGTTAT---3’
O2-647-loop-27
5’—CCGACCGCAGGATCCTATAAAACCCCAACCCC—3’
O1-546 Cell
5’—Alexa-546 -- CCCCAACCCCAATACATTTATATATATCCTAG—3’
O3-cell 5’---TTATAGGATCCTGCGGTCGGACTAGGATATATATAAATGTA---3’
ssDNA 5’-Biotin--AAAAGACTCAC TGTTTGTCTGTCGTTCTAGGATATATAT-3’
ssDNA′ 5’-ATATATATCCTAGAACGACAGACAAACAGTGAGTC-3’
Region 1 5’-ATATATATCCTAG-3’
Region 2 5’-CGACAGACAAACA-3’
Region M 5’-CCTAGAACGACAG-3’
I comp 5’-ATATATATCCTAGAACGACAGACAAACAGTGAGTCCGCATTGTTACAT-3’
I comp′ 5’-ATGTAACAATGCGGACTCACTGTTTGTCTGTCGTTCTAGGATATATAT-3’
Alexa 647 Alexa 647
Alexa647
© 2013 Macmillan Publishers Limited. All rights reserved.
38
Supplementary Table S2. Comparative analysis of organelle acidity measured by
DNA nanodevices either alone or simultaneously, along with literature reported.
Compartments IFuA546/A647 Tf-ITfA488/A647 Reporteda Reportedb
Single SimpHony Single SimpHony
SE 5.98 ± 02 6.0±0.05* 6.09±0.01 6.09± 0.09 6.1(2) 6.2 ± 0.1 (3)
LE 5.72± 08 5.43 ± 0.19 --------------------------------- 5.8 ± 0.1 (4) 5.2-5.8 (5)
RE ------------------------------------- 6.35±0.04 6.56± 0.11 6.5 (2) 6.43 ± 0.03 (6)
TGN 6.18±0.01 6.16 ± 0.09 --------------------------------- 5.95 ± 0.03 (7) 6.19 (8)
* measured by simultaneous pulsing
a,b literature values were chosen from the experiments performed in cells derived from
CHO origin.
© 2013 Macmillan Publishers Limited. All rights reserved.
39
Supplementary Table S3. Comparison of organelle acidity pre- and post-
treatment with brefeldin A
Compartments IFuA546/A647 Tf-ITfA488/A647 IFuA546/A647 Tf-ITfA488/A647
-BFA +BFA
SE/EE --------- 6.07 ± 0.07 ------- 6.14 ± 0.01
Vesicles 5.89±0.16 --------- 6.02 ± 0.09 --------
Tubules ---------- ----------- 6.35 ± 0.17 -------
TGN 5.94 ± 0.15 -------- -------- --------
© 2013 Macmillan Publishers Limited. All rights reserved.
40
Supplementary Movie S1
Dynamics of sorting endosomes in IA2.2 cells after labelling with Tf-ITf alone
IA2.2 cells expressing scFv-Furin were incubated for 1 h in Opti-MEM followed by
labelling of sorting endosomes with a brief pulse of Tf-ITfA488/A647 for 10 min at 37oC.
Images were acquired every 2 s over a 2-3 min time period and compressed in a
movie with 7 fps.
Supplementary Movie S2
Abolition of endosomal motility and fission in dynasore treated IA2.2 cells after
labelling with Tf-ITf alone
IA2.2 cells expressing scFv-Furin were incubated for 1 h in Opti-MEM followed by
labelling of sorting endosomes with a brief pulse of Tf-ITfA488/A647 for 10 min at 37oC.
Dynasore (160 µM) was added to the medium and incubated for 10 min to arrest
fission of sorting endosomes. Images were acquired every 2 s over a 2-3 min time
period and compressed in a movie with 7 fps.
Supplementary Movie S3
Restoration of endosomal motility and fission in cells after labelling with Tf-ITf
and dynasore washout
IA2.2 cells expressing scFv-Furin incubated for 1 h in Opti-MEM followed by
labelling of sorting endosomes with a brief pulse of Tf-ITfA488/A647 for 10 min at 37oC.
Dynasore (160 µM) was added to the medium and incubated for 10 min to arrest
fission of sorting endosomes. Cells were then washed 3-5 times with M1 buffer and
incubated for an additional 10 min at 37oC in complete media to remove dyanasore.
Images were acquired every 2 s over a 2-3 min time period and compressed in a
movie with 7 fps.
Supplementary Movie S4
© 2013 Macmillan Publishers Limited. All rights reserved.
41
Dynamics of sorting endosomes in IA2.2 cells after simultaneous pulse with a
mixture of Tf-ITfA488/A647 and IFu
A546/A647
IA2.2 cells expressing scFv-Furin were incubated for 1 h in Opti-MEM followed by
labelling of sorting endosomes with a brief pulse of Tf-ITfA488/A647 and IFu
A546/647 for 10
min at 37oC. Images were acquired every 2 s over a 2-3 min time period and
compressed in a movie with 7 fps.
Supplementary Movie S5
Abolition of endosomal motility and fission in dynasore treated IA2.2 cells
previously labelled with a simultaneous pulse of Tf-ITfA488/A647 and IFu
A546/A647
IA2.2 cells expressing scFv-Furin were incubated for 1 h in Opti-MEM followed by
labelling of sorting endosomes with a brief pulse of Tf-ITfA488/A647 and IFu
A546/A647 for
10 min at 37oC. Dynasore (160 µM) was added to the medium and incubated for 10
min to arrest fission of sorting endosomes. Images were acquired every 2 s over a 2-3
min time period and compressed in a movie with 7 fps.
© 2013 Macmillan Publishers Limited. All rights reserved.
42
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