doi: 10.1038/nnano.2013.92 two dna nanomachines map ph ... · multiplexing dna nanomachines map ph...

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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


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


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


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).


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


7

spectra were acquired from 500 nm/555 nm to 730 nm by exciting at 488 nm and 546

nm respectively.


8


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


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.


10


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.


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


12


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


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


14


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).


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


16

Individual cells have been demarcated by a white outline. Scale bar: (a) 5 µm (b) 2

µm and (c) 10 µm.


17


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.


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


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.


20


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


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


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


23


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


24


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


25

TGN

EE

SimpHonyA488 FRET647

A546 FRET647


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.


26


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


27


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


28

IFuTfn568

a

Tfn568 EGFP-Furin

b


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.


29


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


30

IFu

IFu

TGN46

TGN46

-BFA

+BFA

*

a

b


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.


31


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


32


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


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.


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


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.


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


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


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.


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 -------- -------- --------


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.


Abolition of endosomal motility and fission in dynasore treated IA2.2 cells after

labelling with Tf-ITf alone



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.


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


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.



41

Dynamics of sorting endosomes in IA2.2 cells after simultaneous pulse with a

mixture of Tf-ITfA488/A647 and IFu

A546/A647


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.


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


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.


42

Supplementary References

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recombinant antibodies as sensors of rab protein conformation. Meth. Enzymol. 403,

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2. Presley, J.F., Mayor, S., McGraw, T.E., Dunn, K.W. & Maxfield, F.R.

Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk

membrane recycling. J. Biol. Chem. 272, 13929–13936 (1997).

3. Yamashiro, D.J. & Maxfield, F.R. Kinetics of endosome acidification in mutant

and wild-type Chinese hamster ovary cells. J. Cell Biol. 105, 2713–2721 (1987).

4. Yamashiro, D.J. & Maxfield, F.R. Acidification of morphologically distinct

endosomes in mutant and wild-type Chinese hamster ovary cells. J. Cell Biol. 105,

2723–2733 (1987).

5. Mayor, S., Presley, J.F. & Maxfield, F.R. Sorting of membrane components from

endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

J. Cell Biol. 121, 1257–1269 (1993).

6. Presley, J.F. et al. The End2 mutation in CHO cells slows the exit of transferrin

receptors from the recycling compartment but bulk membrane recycling is unaffected.

J. Cell Biol. 122, 1231–1241 (1993).

7. Demaurex, N., Furuya, W., D’Souza, S., Bonifacino, J.S. & Grinstein, S.

Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements

of pH using retrieval of TGN38 and furin from the cell surface. J. Biol. Chem. 273,

2044–2051 (1998).


43

8. Maeda, Y., Ide, T., Koike, M., Uchiyama, Y. & Kinoshita, T. GPHR is a novel

anion channel critical for acidification and functions of the Golgi apparatus. Nat. Cell

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doi: 10.1038/nnano.2013.92 two dna nanomachines map ph ... · multiplexing dna nanomachines map ph...

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