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2011 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2011.01215.x

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In situ Measurement of the Electrical Potential Across

the Lysosomal Membrane Using FRET
Mirkka Koivusalo , Benjamin E. Steinberg ,
David Mason and Sergio Grinstein
Program in Cell Biology, Hospital for Sick Children,
Toronto, ON, Canada M5G 1X8
*Corresponding author: Sergio Grinstein,
sergio.grinstein@sickkids.ca
These authors contributed equally to this work.
The progressive acidification of the endocytic pathway
is generated by H+ pumping of electrogenic vacuolartype ATPases (V-ATPases) on the endosomal/lysosomal
membrane. The determinants of pH during endosome
maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect
of the V-ATPase has been proposed to play a central role.
If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the
proton-motive force (pmf ), diminishing the pH gradient
proportionally. Validation of this notion requires direct
measurement of the electrical potential that develops
across the endosomal/lysosomal membrane. To date,
the measurement of lysosomal membrane potential ( )
in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that
individual organelles cannot be discerned when using
potentiometric fluorescent dyes. Here, we describe a
noninvasive procedure to estimate in intact cells,
based on fluorescence resonance energy transfer (FRET).
At steady state, averaged 19 mV (lumen positive)
and was only partially dissipated by inhibition of the
V-ATPase with concanamycin A (CcA). was considerably increased by alkalinization of the lysosome lumen by
NH4 Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of
to pmf is relatively small. Our method should enable
systematic studies of endosomal/lysosomal potential.
Key words: electrical potential, FRET, lysosome, protonmotive force
Received 1 January 2011, revised and accepted for publication 3 May 2011, uncorrected manuscript published
online 6 May 2011, published online 1 June 2011

During endocytosis, the internalized cargo encounters a

progressively acidic pH as it proceeds to degradative
lysosomes. The proper acidification of endosomes and
lysosomes plays a crucial role in the regulation of several
important cellular functions, including receptor-ligand dissociation, vesicular trafficking, hydrolysis of internalized
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macromolecules and assembly of coat proteins on the

organellar surface (reviewed in 1,2). While it is clear that
the existence of a gradual transmembrane pH gradient
is central to endocytic function, the mechanism whereby
the progressive acidification is established remains largely
unknown.
The acidic pH of endosomes and lysosomes is generated
by vacuolar-type ATPases (V-ATPases), which transport
protons at the expense of ATP. Because the V-ATPase is
an electrogenic pump, a transmembrane electrical potential (lumen positive) tends to develop when protons are
translocated into the compartment. The developing voltage limits further pumping, and significant net proton
translocation requires dissipation of the electrical potential
by parallel counter-ion fluxes (24). In the presence of permeable counter ions, the emerging pH gradient together
with the residual electrical potential combines to establish
a passive proton-motive force (pmf ) that antagonizes the
action of the V-ATPase. A differential contribution of the
electrical and chemical components to the pmf has been
suggested as one of the mechanisms to account for the
progressive acidification of the endocytic pathway. Thus,
an increasing counter-ion conductance that more effectively dissipates the electrical component would account
for the greater acidification of lysosomes (3,5,6). However, gradations in pH could alternatively be established
by varying degrees of H+ leakage, which would dissipate
the forming pH gradients to different extents (3,4,6,7).
Differentiation between these models of endosome acidification requires that the contribution of membrane potential to the pmf can be accurately measured. There are
well-established methods for in situ pH measurement of
endocytic organelles but it has been virtually impossible to
measure the electrical potential across membranes of the
endocytic pathway. Traditionally, electrical potential across
the plasma membrane is measured using electrophysiological techniques. Measurement of membrane potential across intracellular membranes by electrophysiology,
however, is limited by the small size of organelles and their
inaccessibility to electrodes. Membrane-permeable potentiometric probes can also be used to measure potential
across membranes. Indeed, potentiometric fluorescent
dyes have been used to estimate the membrane potential
of isolated endosomes and lysosomes (3,5,6,8). However,
using these dyes in intact cells is not feasible, because
they partition across all cellular membranes, making it
impossible to resolve the fluorescence contribution of individual compartments. Here, we describe a novel method
employing fluorescence resonance energy transfer (FRET)

Lysosomal Membrane Potential

to measure in situ the steady-state membrane potential

across the lysosomal membrane in live cells. The method
is based on the targeted delivery of a fluorescent lipid
analog to lysosomes, followed by addition of an anionic,
lipid-soluble potentiometric oxonol dye.

Results
Characterization of the FRET probes
We developed two different FRET-based systems to
measure membrane potential in lysosomes. In the first
approach, a member of the oxonol family of potentiometric dyes, bis-(1,3-dibutylbarbituric acid)trimethine oxonol
(DiBAC4 (3)), was selected as the FRET donor and phosphatidylethanolamine labeled in the head group with
a rhodamine derivative, L--phosphatidylethanolamine-N (lissamine rhodamine B sulfonyl) or Rh-PE, as the FRET
acceptor (Figure 1A). The uncorrected fluorescence spectra of the two dyes, illustrated in Figure 1B, show that
the overlap between donor (DiBAC4 (3)) emission and
acceptor (Rh-PE) excitation was substantial (Figure 1B).
To validate the occurrence of FRET between the dyes,
CH3(CH2)3 OH

N
O

B 1.2

(CH2)3CH3

Donor ex
Donor em
Acceptor ex
Acceptor em

N
CH

CH

CH

CH3(CH2)3 O

(CH2)3CH3

Donor: DiBAC4(3)
N

In the second approach, we synthesized the FRET

donor by covalently linking the coumarin derivative
7-diethyaminocoumarin-3-carboxylic acid succinimidyl
ester (DACCA-SE) to the head group of phosphatidylethanolamine (DACCA-PE; Figure 2A), followed by
purification of the conjugate by column chromatography.
The purity of the product was verified by thin-layer
chromatography (TLC). Another oxonol dye, bis-(1,3diethylthiobarbituric acid)trimethine oxonol (DiSBAC2 (3)),
was selected as the FRET acceptor (Figure 2A). Spectrofluorimetric analysis of the dyes again showed a substantial overlap between the donor emission and acceptor
excitation spectra (Figure 2B). To assess whether FRET

Fluorescence (a.u.)

a constant amount of DiBAC4 (3) was titrated with eggphosphatidylcholine (PC) vesicles containing increasing
concentrations of Rh-PE. The emission spectrum was
recorded using excitation of the donor at 480 nm; as
illustrated in Figure 1C the peak emission of the donor
decreased and acceptor emission appeared and increased
as the concentration of Rh-PE increased, consistent
with energy transfer between these two fluorophores
(Figure 1C).

SO3

O S

N
H

O-

0.6
0.4
0.2

O P O

0.8

O
H

450

Acceptor: Rhodamine-PE

500

550

600

650

Wavelength (nm)

C 1.2

0 nM
10 nM
25 nM
50 nM
100 nM
Acceptor alone

Fluorescence (a.u.)

1
0.8
0.6
0.4
0.2

500

550
600
Wavelength (nm)

650

Figure 1: Characterization of DiBAC4 (3) and Rh-PE as a FRET donor and acceptor, respectively. A) The structure of DiBAC4 (3) and
Rh-PE. B) Excitation (blue) and emission spectra (green) of DiBAC4 (3) in the presence of egg-PC liposomes. Excitation (yellow) and
emission spectra (red) of 0.5% Rh-PE in egg-PC liposomes. The gray bars illustrate the filter bandwidths used for visualization of FRET
by microscopy. C) Emission spectra obtained in the presence of both DiBAC4 (3) and Rh-PE with excitation at 480 nm. Spectra obtained
using 300 nM DiBAC4 (3) and the indicated concentrations of the acceptor (Rh-PE, 0100 nM, corresponding to 00.5% of total lipid) in
egg-PC liposomes are illustrated.

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Koivusalo et al.
(CH3CH2)2N

O
O

N
O

o/n, shaking

DACCA succinimidyl ester

(CH3CH2)2N

+
O
H 2N

O P O

N
H

O-

O
H

O
O

O P O
O-

O
H

Donor: DACCA-PE

O
O

PE
CH3CH2

OH

CH2CH3

CH

CH

CH

N
O

CH3CH2

CH2CH3

Acceptor: DiSBAC2(3)

Fluorescence (a.u.)

Donor ex
Donor em
Acceptor ex
Acceptor em

1
0.8
0.6
0.4
0.2

350

C
1.2
Fluorescence (a.u.)

1.2

0 nM
100 nM
200 nM
300 nM
400 nM
500 nM
Acceptor alone

1
0.8
0.6
0.4
0.2

400

450
500
550
Wavelength (nm)

600

650

400

450

500
550
Wavelength (nm)

600

Figure 2: Characterization of DACCA-PE and DiSBAC2 (3) as a FRET donor and acceptor, respectively. A) Schematic of the
synthesis of DACCA-PE; also shown is the structure of DiSBAC2 (3). B) Excitation (solid purple) and emission spectra (dotted blue) of
DACCA-PE in egg-PC liposomes containing 1% DACCA-PE. Excitation (solid green) and emission spectra (dotted yellow) of DiSBAC2 (3) in
the presence of egg-PC liposomes. The gray bars illustrate the filter bandwidths used for visualization of FRET by microscopy. C)
Emission spectra obtained in the presence of both 200 nM (1% of total lipid) DACCA-PE in egg-PC liposomes and DiSBAC2 (3) with
excitation at 400 nm; individual spectra were obtained at the acceptor (DiSBAC2 (3)) concentrations indicated (0500 nM). Note that in
the absence of donor molecules there was no detectable fluorescence, even at 1 M DiSBAC2 (3).

can occur between these dyes, DACCA-PE was incorporated into egg-PC liposomes that were titrated with
increasing amounts of DiSBAC2 (3), while recording the
emission spectrum upon excitation of the donor at 400 nm
(Figure 2C). As the amount of acceptors increased, the
donor emission progressively decreased while the acceptor emission increased. Although not as marked as in the
case of DiBAC4 (3) and Rh-PE (Figure 1C), the magnitude
of the FRET between DACCA-PE and DiSBAC2 (3) was
nevertheless substantial.

Targeting FRET probes to the lysosomal

compartment in RAW264.7 macrophages
Because the potentiometric oxonols are soluble and membrane permeant, they distribute throughout the cells and
are unable to provide organelle-specific information. To
selectively measure lysosomal membrane potential, we
therefore used energy transfer, targeting the paired fluorophores to the compartment of interest. In the case
of DiBAC4 (3), employed as a fluorescence donor, Rh-PE
was used as an acceptor, whereas DACCA-PE was used
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as a donor for DiSBAC2 (3). PE derivatives with covalent

labels attached to their head-group were described earlier to be sorted to lysosomes after internalization (9,10).
We therefore devised protocols to selectively target the
fluorescent phosphatidylethanolamine derivatives to lysosomes, using RAW264.7 cells as a model system. These
murine macrophages perform active constitutive endocytosis, expediting the labeling procedure. We used Alexa
488-dextran to identify lysosomes in intact cells. A 20-h
incubation with this marker ensured preferential accumulation in lysosomes, and residual label in endosomes was
forwarded to lysosomes during the course of the subsequent delivery of the labeled lipid. A fraction of the
label may remain in late endosomes under these conditions, but it is likely very small and will be disregarded
hereafter. After Alexa 488-dextran labeling, the cells were
incubated with Rh-PE complexed to BSA for 10 min on ice,
to allow incorporation of the lipid into the plasma membrane. The label was then chased at +37 C and imaged
at different times. Imaging by spinning disc confocal
fluorescence microscopy showed that shortly after labeling Rh-PE was predominantly in the plasma membrane but

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Lysosomal Membrane Potential

10 min

Marker

60 min

Rhod-PE

Alexa488-dextran

Rh-PE

Overlay

Overlay

Sig seqGFP-KDEL

GalT-GFP

Mitotracker

DQ-BSA

Rab11A-GFP

Rab5-GFP

30 min

DACCA-PE

TMR-dextran

Overlay

Figure 3: Distribution of Rh-PE and DACCA-PE in live RAW264.7 macrophages. A) Cells were labeled with Rh-PE as described in
Materials and Methods and chased for the indicated times before acquiring confocal images using spinning disc microscopy. In the two
right-most panels, the lysosomes were prelabeled with Alexa 488-dextran and the localization relative to Rh-PE chased for 60 min is
illustrated. B) Comparison of the localization of different organellar markers with Rh-PE chased into cells for 6090 min, as described
in Materials and Methods. The right-most panels show an enlargement of the region delineated in the adjacent overlay panels. C)
Localization of DACCA-PE after a 60-min chase, compared to TMR-dextran-labeled lysosomes. Scale bars = 10 m.

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Koivusalo et al.

gradually moved to punctate structures which, by 60 min,

colocalized with lysosomal dextran (Figure 3A). To better
characterize the compartment where Rh-PE resided after
60 min, we compared its localization with that of various
organellar markers (Figure 3B). At this stage, Rh-PE did
not show significant colocalization with the early endosomal marker Rab5-GFP or the recycling endosomal marker
Rab11A-GFP. Conversely, Rh-PE colocalized extensively
with DQ-BSA, a probe that becomes fluorescent only
in the proteolytic environment of lysosomes. Neither
the Golgi marker galactosyltransferase-GFP (GalT-GFP),
the mitochondrial marker Mitotracker, nor an endoplasmic reticulum marker (GFP with the signal sequence of
preprolactin and a C-terminal KDEL retention sequence)
colocalized with Rh-PE, as expected (Figure 3B).
The concentration of Rh-PE attained in lysosomes using
this procedure was estimated in parallel experiments measuring the fluorescence of lysates of loaded cells prepared
in PBS containing 1% Triton-X-100 and comparing them
to equivalent lysates from unlabeled cells mixed with
known amounts of Rh-PE, and in parallel measuring the
volume occupied by lysosomes in the cells. The latter was
accomplished by optical sectioning and three-dimensional
reconstruction of images of cells, where lysosomes had
been loaded with rhodamine-dextran (see Materials and
Methods for details). Using this approach, we estimate
the concentration of Rh-PE in lysosomes to be 135 M.
To investigate DACCA-PE localization in RAW264.7 cells,
this lipid analog was introduced to the plasma membrane
using methyl--cyclodextrin (mCD)-mediated transfer.
After pulse labeling the cells for 5 min followed by a
1-h chase, imaging by spinning disc confocal fluorescence
microscopy showed that, as found for Rh-PE, DACCA-PE
was in punctate structures that largely colocalized
with dextran previously chased into the lysosomal
compartment (Figure 3C). These observations indicate
that, with the labeling protocol used, both lipid analogs
can be reliably and consistently targeted to lysosomes.

Imaging FRET in live RAW264.7 macrophages

To measure FRET in macrophages, cells were first labeled
with fluorescent PE analogs followed by addition of the
membrane-permeant oxonol dye to the bathing medium.
Oxonols were allowed to equilibrate across all cellular
membranes and epifluorescence microscopy was used to
sequentially record the emission in the donor, acceptor
and FRET channels. Bleed-through of donor and acceptor
fluorescences was subtracted from the FRET readings
to calculate the corrected fluorescence resonance energy
transfer (cFRET; see Materials and Methods for details).
Cells labeled with donor alone, whether DiBAC4 (3) in the
case of the first FRET pair (Figure 4A) or DACCA-PE in the
second case (Figure 4B), showed negligible cFRET signal. Similarly, cFRET was virtually absent when only the
acceptor, Rh-PE in one case (Figure 4A) or DiSBAC2 (3) in
the other (Figure 4B), was present. Note also that both
potentiometric oxonol dyes were diffusely distributed
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throughout the cell, precluding visual delineation of individual organelles. Importantly, in macrophages that had
been labeled with both the donor and the acceptor, a
cFRET signal was clearly detectable and restricted to the
punctate lysosomes (Figure 4A,B).

Lysosomal membrane potential ( ) measurement

Having confirmed the effectiveness of the FRET pairs, we
proceeded to measure the lysosomal membrane potential
( ). The cFRET signal, a function of the oxonol concentration in the immediate vicinity of the lysosomal PE probe,
is an indirect measure of and must be calibrated to
obtain quantitative estimates. We devised an in situ calibration protocol to calculate the magnitude of from
the cFRET values. The rationale applied to calibrations
is similar to the one used before (11) and is illustrated
schematically in Figure 5A. The system consists of three
compartments: the extracellular space (compartment 1),
the cytosol (compartment 2) and the intralysosomal compartment (compartment 3). An electrical potential difference PM exists across the plasma membrane, which
separates extracellular space and cytosol, whereas a distinct membrane potential is assumed to exist across
the lysosomal membrane. The concentration of the oxonol
in each compartment (Cx ) is dictated by PM and . For a
given extracellular oxonol concentration C1 , the cytosolic
concentration C2 is a function of PM . The intralysosomal
oxonol concentration C3 , on the other hand, is dependent
on both C2 and . The relationships between the five
variables can be described by the following equations:


z FPM
C2 = C1 exp
(1)
RT
 
C2
RT
(2)
log
=
zF
C3
where R, T and F are the gas constant, temperature in
Kelvin and Faradays constant, respectively, and z = 1,
the charge of the oxonol dyes. As a result, can be
calculated if the values for C2 and C3 are defined.
We determined the value for C3 from external calibrations performed separately for each experiment by
using DiBAC4 (3) and Rh-PE as the FRET pair. To this
end, RAW264.7 cells labeled with Rh-PE were incubated in K+ -rich buffer, which induces depolarization of
the surface membrane. The monovalent cationophore
gramicidin A was included in the medium to aid in equilibrating K+ across cell membranes and concanamycin
A (CcA) to inhibit the contribution of V-ATPase to the
lysosomal membrane potential. Different concentrations
of the oxonol DiBAC4 (3) were added to the buffer, allowed
to equilibrate across the cellular membranes and cFRET
measured in lysosomes. Under these conditions, the
potential across both the surface and lysosomal membranes is assumed to be negligible and the extracellular
concentration of the oxonol should be virtually identical
to that in the cytosol and in the lysosomal lumen. The
intensity of the cFRET signal also depends on the concentration of Rh-PE. It is important to note that calibrations

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Lysosomal Membrane Potential

DIC

DiBAC4(3)

Rhod-PE

cFRET

DIC

DACCA-PE

DiSBAC2(3)

cFRET

Donor & Acceptor

Acceptor alone

Donor alone

Donor & Acceptor

Acceptor alone

Donor alone

were always performed for each individual experiment

using cells labeled at the same time; this ensured the
Rh-PE (acceptor) concentration would be identical for the
experimental and calibration samples. This was validated
experimentally by comparing the fluorescence of Rh-PE in
parallel samples exposed to varying concentrations of the
donor, DiBAC4 (3) (Figure S1).
The measurements obtained using increasing concentrations of DiBAC4 (3) were used to generate a calibration
curve (Figure 5B) that enabled us to interpolate C3 from the
cFRET values of experimental determinations. By using
eqn (1), we calculated C2 from the known value of C1

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Figure 4: FRET imaging in live

RAW264.7 macrophages. A) Cells
labeled with only donor 300 nM
DiBAC4 (3) (top row), only acceptor RhPE (middle row), or both the donor and
the acceptor (bottom row). For each
condition, epifluorescence microscopy
images were sequentially acquired in the
donor, acceptor and cFRET channels.
The corresponding differential interference contrast (DIC) images are also
illustrated. B) Cells labeled with only
donor DACCA-PE (top row), only acceptor 125 nM DiSBAC2 (3) (middle row) or
both the donor and the acceptor (bottom row) and imaged as in (A). Scale
bars = 10 m.

and using a previously determined value of 69.1 mV for

PM in resting RAW264.7 cells (11). From the ratio of C3
and C2 we were able to calculate the lysosomal membrane potential, . In otherwise untreated RAW264.7
cells, the average steady-state was estimated to be
18.6 1.3 mV (lumen positive; mean SE of seven
experiments measuring at least 35 lysosomes in each;
Figure 5C). Very similar results were obtained using two
different DiBAC4 (3) concentrations, 150 or 300 nM, supporting the reliability of the determinations.
It is noteworthy that the calculated luminal concentration
of DiBAC4 (3) (89 7.6 nM under basal conditions when
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Koivusalo et al.
Lysosome ()

B 3000
Extracellular
space

2500

C1
cFRET

2000

C3
Cytosol

1500
1000

C2

500

PM
PM

50
100
[DiBAC4(3)] nM

150

35

***

30

6
5.5
5

20

pH

mV

***

6.5

25

6.5
pH

4.5

5.5

5
15
Min after CcA

30

15

**

10

5
4.5

4
Basal

Basal

NH4Cl

NH4 Cl

CcA

Basal

NH4Cl

CcA

CcA

Figure 5: Lysosomal membrane potential measurement with FRET. A) Variables for the calculation of . The system comprises
the extracellular (compartment 1), intracellular (compartment 2) and intralysosomal (compartment 3) spaces. The concentration of
free DiBAC4 (3) in each compartment is indicated as Cx , where x denotes the compartment. Compartments 1 and 2 are separated
by the plasma membrane and compartments 2 and 3 by the lysosomal membrane, with transmembrane potentials PM and ,
respectively. B) External calibration used to calculate C3 . RAW264.7 cells were incubated with gramicidin (1 M) during the Rh-PE
chase, and then transferred into K+ -rich buffer containing 1 M gramicidin and 1 M CcA to collapse the membrane potentials. Different
concentrations of DiBAC4 (3) were added and the amount of cFRET was measured within the lysosomes. The amount of cFRET was
then translated into C3 by using the calibration curve determined separately for each experiment. A representative calibration curve of
cFRET versus DiBAC4 (3) concentration (C1 ) is shown. Data are means of 6595 lysosomes SD. C) Measurement of basal and
5 min after addition of 20 mM NH4 Cl or 1 M CcA, as indicated, measured by using 300 nM or 150 nM DiBAC4 (3) in the bathing
medium (data obtained at both concentrations were similar and were grouped together). Data are means of five to seven experiments
SEM with at least 35 lysosomes in each. p < 0.001, p < 0.01 (ANOVA with a Dunnets test), relative to the basal condition. D)
Measurement of lysosomal pH under the same conditions as in (C), obtained by ratiometric imaging of lysosomes loaded with a mixture
of FITC-dextran/OG488-dextran, as described in Materials and Methods. The inset illustrates the progressive alkalinization of lysosomes
at the indicated times after addition of 1 M CcA. Data are means of three to eight experiments SEM with 10 lysosomes in each.
p < 0.001 (ANOVA with a Dunnets test), relative to the basal condition. E) Representative cFRET images for each condition. Scale
bar = 10 m.

the extracellular concentration was 300 nM) is comparatively low. At face value, this amount of dye would be
expected to yield little fluorescence. However, the partition coefficient of DiBAC4 (3) greatly favors hydrophobic
environments (12); therefore, the concentration of the
dye within the lysosomal membrane is several orders
978

of magnitude higher than that in the aqueous phase of

the lumen, clearly sufficient for detection. Indeed, this
principle guided our choice of Rh-PE which is located in
the bilayer as an acceptor. We tested earlier the soluble rhodamine-dextran as an acceptor and obtained much
weaker cFRET signals (Figure S2).

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Lysosomal Membrane Potential

Contribution of the V-ATPase to

Next, we assessed the contribution of the electrogenic
V-ATPase to , as it is the main contributor to lysosome acidification. Proton pumping by the V-ATPase
was arrested by addition of maximally inhibitory concentrations of the specific inhibitor CcA, and 5 min later
cFRET was measured in lysosomes as above. Inhibition
of the V-ATPase decreased the cFRET values significantly (Figure 5E). Calculation of the membrane potential
revealed a decrease of 50%, to 9.4 0.9 mV (lumen
positive; Figure 5C), indicating that the electrogenic activity of the pump is an important contributor to the lysosomal
membrane potential at steady state. However, other components exist; a Donnan potential is a likely contributor to
the residual voltage. It is noteworthy that at the time when
was measured, i.e. 5 min after addition of CcA, the
change in lysosomal pH was negligible (Figure 5D). The
low rate of H+ (equivalent) leakage together with the large
buffering power of the lysosomes accounts for this observation. Indeed, the dissipation of the transmembrane H+
gradient proceeded gradually for the following 30 min
(see inset in Figure 5D). Lysosomes are rich in proteins,
as evinced by their electron density, and many of these
are likely to bear a net positive charge, considering the
low prevailing pH. This would favor the generation of a
sizable Donnan potential.
The existence of an electrical potential together with a
sizable transmembrane pH gradient implies that a considerable pmf opposes the activity of the V-ATPase. As
such, it is likely that the enzyme is not operating at its
full capacity. To test this notion, we partially relieved the
pmf by alkalinizing the lysosomal pH. This was accomplished by addition of NH4 + , a membrane-permeant weak
base. Independent lysosomal pH measurements verified
that addition of 20 mM NH4 Cl to the solution bathing the
cells induced a large and sustained alkalinization, from
pH 4.9 to 6.6 (Figure 5D). Concomitantly, the accumulation of oxonol measured by cFRET increased markedly
(Figure 5C,E). Calibration of this enhanced signal yielded
an average membrane potential of 28.5 1.2 mV (lumen
positive; Figure 5C). This hyperpolarization was abolished
when cells were (pre)treated with CcA. When both the
inhibitor and NH4 Cl were present simultaneously, the
potential dropped to 10 mV, indistinguishable from that
observed in the presence of CcA alone (not shown).
Similar results were obtained when using the other
FRET pair, DACCA-PE and DiSBAC2 (3): decreased
upon addition of CcA and increased with NH4 Cl (not
illustrated). Although producing less intense cFRET, the
results obtained with DACCA-PE and DiSBAC2 (3) offer
independent confirmation of the observations made with
DiBAC4 (3) and Rh-PE. In addition, HeLa cells responded
similarly to these two treatments (not shown), indicating
that the effects were not unique to RAW264.7 cells and
that the method is applicable to other cell types.

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Discussion
To our knowledge, this is the first method described to
measure lysosomal membrane potential in live cells. FRET
had been used previously to measure the plasmalemmal
membrane potential (13,14), but intracellular organelles
present unique challenges on account of their inaccessibility and much smaller size. We estimated that lysosomes
at rest have a lumen-positive of nearly 20 mV, which
is of the same order as previously suggested values for
other endocytic compartments. In live cells, potentials of
1020 mV were calculated from measurements of chloride concentration (15), based on the premise that chloride
permeability is the dominant contributor to the endosomal conductance and that chloride is at electrochemical
equilibrium; these assumptions need to be validated. The
membrane potential was also measured previously in isolated endosomes; potentials of 2030 mV were obtained
in buffers containing Cl concentrations resembling that of
the cytosol (6). Lastly, we recently reported that the membrane potential of another endocytic organelle, the phagosome, is similarly positive inside, averaging 27 mV (11). All
these organelles share one feature in common, namely
the presence of active electrogenic H+ pumps. In the case
of lysosomes, we found that a fraction of the steady-state
membrane potential is directly attributable to the activity
of the V-ATPases.
By combining the membrane potential determinations presented here with independent measurements of the pH
gradient (16), we can estimate directly the pmf across
the lysosomal membrane. In RAW264.7 cells, the lysosomal and cytosolic pH are 4.64 and 7.37 (16), respectively,
resulting in a pH of 2.73 units across the lysosomal membrane. From these measurements, the pmf across the
lysosomal membrane was calculated to reach 18.0 kJ/mol
at steady state, 1.8 kJ/mol of which is contributed by the
electrical component. This antagonizing force curtails the
activity of the V-ATPase, which operates at submaximal
rate in the steady state. This became apparent when the
pH was reduced by addition of NH4 + , which resulted in
a CcA-sensitive hyperpolarization. Yet, while significant,
the measured pmf is considerably lower than the theoretical limit imposed by the energy available from ATP
hydrolysis, the driving force of the V-ATPase. Assuming a
stoichiometry of 2 H+ per ATP hydrolysis cycle, a theoretical limit of approximately 29 kJ/mol is available to generate
the lysosomal electrochemical H+ gradient (17,18). That
the pmf estimated empirically falls short of this value
implies imperfect coupling. At least two mechanisms can
be readily envisaged to account for this: first, the energy
conversion efficiency of the V-ATPase is <100%. Second,
the lysosomal membrane is known to harbor one or more
H+ leak pathways, which allow for futile cycling between
active H+ influx and passive efflux (3,4,6,7). Our measurements of lysosomal pH following addition of CcA confirm
the presence of the leak pathway (inset, Figure 5D).
979

Koivusalo et al.

The absolute magnitude of the membrane potential is

likely to vary along the endocytic pathway. If the pmf
were to stay constant across compartments, one would
anticipate a more positive potential at earlier stages, which
would decline as the luminal acidification increases. The
balance between the chemical and electrical components
of the pmf is presumably dictated by the counter-ion
permeability. That counter ions are required for effective
acidification has been repeatedly shown in vitro (3,5,6,8).
However, such studies fail to identify the ion(s) that are
physiologically relevant. This information is best gleaned
from measurements in situ. In live cells, a parallel Cl
gradient has been shown to be required for the establishment of a pH gradient in endosomes (15,19). In contrast,
Cl is not absolutely required as a counter ion to acidify
lysosomes in situ; efflux of luminal cations can equally sustain lysosome acidification (16). The additional counter-ion
pathway(s) possibly enable the lysosome to more readily
compensate the electrogenicity of the V-ATPase, enabling
it to acidify the lysosomal lumen more profoundly.
While having provided the first direct measurements of
lysosomal membrane potential, we regard the main value
of this report as being technical in nature. We believe that
the FRET-based assay introduced here to determine lysosomal membrane potential can be modified and extended
to measure the membrane potential of other endocytic
and secretory compartments that to date have remained
inaccessible by other approaches. A variant of this procedure could be used to test the role of the electrogenic
Na,K-ATPase in early endosomes, where it purportedly
limits acidification by increasing membrane potential (20).
Similarly, the operation of other ion channels, transporters
and pumps residing within the endocytic pathway represented could be similarly probed. Such measurements
would improve our understanding of ion transport and pH
homeostasis in endomembranes.

Materials and Methods

Reagents
DACCA-SE, DiBAC4 (3), DiSBAC2 (3), DQ-BSA green, Mitotracker green,
nigericin and tetramethylrhodamine (TMR)-, rhodamine B-, Alexa 488-, fluorescein isothiocyanate (FITC)- and Oregon Green 488 (OG488)-conjugated
dextrans [all of molecular weight (MW) 10 000] were from Invitrogen. Egg-Rh-PE, egg-PC and 16:0/18:1-PE [palmitoyloleoylphosphatidyl
ethanolamine (POPE)] were from Avanti Polar Lipids. FugeneHD was from
Roche. All other reagents were from Sigma-Aldrich.
Isotonic Na+ -rich buffer contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl2 ,
1 mM CaCl2 , 5 mM glucose and 20 mM HEPES (pH 7.4). In K+ -rich buffer,
NaCl was replaced by 100 mM K-glutamate and 43 mM KCl.

DACCA-PE synthesis
POPE in CHCl3 was combined with 5.5 molar excess of DACCA-SE
in dimethyl sulfoxide, and the mixture incubated overnight with vigorous
shaking. The reaction mixture was dried under N2 , resuspended in 98:2
CHCl3 :MeOH and layered onto a 2-cm silica column in the same solvent.
The column was washed with 98:2 CHCl3 :MeOH until all the unreacted
DACCA-SE had been washed out (5 2 mL). The DACCA-PE was eluted

980

out in consecutive 90:10:0.5 CHCl3 :MeOH:H2 O fractions monitored by

TLC. Fractions containing the end product were used for cell labeling.

FRET pair characterization

To measure the excitation and emission spectra of Rh-PE and DACCA-PE
in the absence and presence of the oxonols, these lipids were incorporated into egg-PC vesicles at different concentrations. Lipids were dried
under N2 and reconstituted into multilamellar vesicles by vortexing to
20 M final lipid concentration in PBS. Excitation and emission spectra
of DiBAC4 (3) and DiSBAC2 (3) were measured in the presence of egg-PC
liposomes without the fluorescent lipids. Spectra were acquired by using
an F-2500 Fluorescence Spectrophotometer (Hitachi).

Cell culture, plasmids and transfections

RAW264.7 macrophages (ATCC number TIB-71) were obtained from the
American Type Culture Collection and grown at 37 C under 5% CO2 in
RPMI-1640 supplemented with 5% FBS (Wisent). The plasmids encoding Rab5A-GFP (21) and Rab11A-GFP (22) have been described elsewhere
and were a kind gift from Dr John Brumell (Hospital for Sick Children,
Toronto). Preprolactin signal sequence-GFP-KDEL has also been previously
described (23). GalT-GFP was from Addgene (plasmid #11929) (24). Transfections were performed with FugeneHD following the manufacturers
instructions.

Labeling of cells with DACCA-PE

DACCA-PE was introduced into RAW264.7 cells using mCD-mediated
transfer (25,26). For labeling one 18-mm coverslip, 36 g DACCA-PE
and 5 g cholesterol were dried under N2 ; 100 mM mCD was added
and the mixture probe sonicated for 3 2 min (0.9 g/L DACCA-PE and
0.125 g/L cholesterol in the mixture). Forty microliters of the mixture
was added to cells in 500-L serum-free RPMI for a final concentration
of 67 and 9.3 g/mL for DACCA-PE and cholesterol, respectively, and
7.4 mM for mCD. The cells were pulsed with the lipid mixture for 5 min
at 37 C, washed and chased for 15 min in serum-free RPMI followed by
a 1560-min chase in RPMI + 5% FBS to remove any residual DACCA-PE
left on the plasma membrane. Cells were then transferred to serum-free
HEPES-buffered RPMI (HPMI) or Na+ -rich buffer for imaging.

Labeling of cells with Rh-PE

Rh-PE was freshly complexed with fatty acid-free BSA by adding 25 L
of 1 mg/mL Rh-PE solution in 1:1 CHCl3 /MeOH dropwise into 4 mL of
0.5 mg/mL BSA in PBS while vigorously vortexing for 12 min. A gentle
stream of N2 was blown on the complex before placing it on ice. RAW264.7
cells grown on 18- or 25-mm coverslips were washed with ice-cold PBS,
and incubated with the Rh-PEBSA complex for 10 min on ice to first
introduce the lipid to the plasma membrane. Cells were washed with
warm HPMI or Na+ -rich buffer, and chased in this buffer at 37 C for
6090 min to label the lysosomes.

Lysosomal membrane potential measurements

A 150 or 300 nM DiBAC4 (3) was added into the bathing medium of RhPE-labeled cells and cells allowed to equilibrate for 5 min at 37 C. For
DACCA-PE-labeled cells, 125 nM DiSBAC2 (3) was added to the bathing
medium and cells equilibrated as above. Fifteen to 30 baseline images of
different fields of the donor, acceptor and FRET channels were acquired.
The cells were then treated with 1 M CcA or 20 mM NH4 Cl, or both. After
5 min of incubation, 1530 images in the three channels were acquired.
For the determination of the donor and acceptor bleed-through coefficients,
samples with donor and acceptor only were imaged in each experiment.
To determine the amount of DiBAC4 (3) within the lysosomes, separate
calibration samples were treated as follows. During chase with Rh-PE,
1 M gramicidin was included in HPMI. Cells were then washed with
K+ -rich buffer and incubated with 25300 nM DiBAC4 (3), 1 M CcA and

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Lysosomal Membrane Potential

1 M gramicidin A in K+ -rich buffer to collapse the membrane potential.
After 5 min of incubation, 1530 images of different fields in all the
three channels were acquired. In this way, a calibration curve relating
cFRET to DiBAC4 (3) concentration was constructed, and the intralysosomal DiBAC4 (3) concentration determined from the linear fit of the data.
To adjust the microscope settings for each experiment, measurements
were started with the highest DiBAC4 (3) concentration in K+ -rich buffer,
which gave the highest DiBAC4 (3) fluorescence inside the cells. was
calculated as described in Results.

a Live Cell Instrument environmental control system. The samples were

excited with diode-pumped solid-state laser lines (Applied Research) using
a 491-nm laser line and 520-nm emission filter for green fluorophores,
a 561-nm laser line and 590-nm emission filter for Rh-PE and a 405-nm
laser line and 457-nm emission filter for DACCA-PE. A 63/NA 1.4 oil
immersion objective was used. Images were acquired using a Hamamatsu
C9100-13 ImagEM back-thinned electron multiplier CCD camera driven by
the VOLOCITY 4.1.1 software (Perkin Elmer).

Estimation of intralysosomal Rh-PE concentration

Live-cell imaging and FRET
Coverslips were mounted in Chamlid imaging chambers (Live Cell Instrument Inc.) and maintained at 37 C on the stage of a Leica DM IRB microscope equipped with filter wheels (Sutter Instruments) to independently
alternate between different excitation and emission filters. Light from
an EXFO X-Cite 120 lamp (Exfo Life Sciences Group) was directed to
the sample by using a dichroic mirror. Emitted light was captured by
a Cascade II CCD camera (Photometrics). The filter wheel and camera were under the control of METAMORPH/METAFLUOR software (Molecular
Devices). In FRET experiments, three fluorescent channels were serially
acquired. The filter set configurations (listed as excitation/dichroic/emission
filters, all in nanometers bandwidth) used in the FRET experiments
were as follows: donor DiBAC4 (3) 475 60/495/510 40, acceptor Rh-PE
543 22/565/580 25 and FRET 475 60/565/580 25; donor DACCA-PE
417 60/425/470 40, acceptor DiSBAC2 (3) 543 22/565/580 25 and
FRET 417 60/565/580 25. A PL Fluotar 100/numerical aperture (NA)
1.3 oil immersion objective was used. cFRET was calculated using the
method of Youvan et al. (27). The donor and acceptor bleed-through coefficients were determined by acquiring images with donor or acceptor alone,
respectively, in independent samples. All microscope parameters, including exposure times and camera gains, were kept constant across each
experiment. Fluorescence intensity measurements were performed using
IMAGEJ software. Images were background-subtracted before analysis.

Cells were loaded with Rh-PE as described above and after a 1-h chase
they were scraped and suspended into PBS containing 1% Triton-X100. The fluorescence emitted by Rh-PE was measured using a Hitachi
spectrofluorimeter. The amount of Rh-PE per cell was estimated by
interpolation in a standard calibration curve obtained adding known
amounts of Rh-PE into a cell lysate prepared from unlabeled cells; the
amount of unlabeled lysate used in this instance was identical to that
used for the loaded cells above. To estimate the volume of lysosomes,
RAW264.7 cells were labeled with 0.2 mg/mL rhodamine B-dextran for
16 h, chased for 1 h and imaged by spinning disc confocal microscopy.
Serial optical slices were collected and used to construct a threedimensional image. The average volume occupied by the lysosomes in
each cell was estimated by using the IMAGEJ (NIH) 3D object counter plug-in.

Acknowledgments
This work was supported by grants from the Canadian Institutes of
Health Research (CIHR grant MOP4665) and the Canadian Cystic Fibrosis
Foundation. M. K. was supported by the Ella and Georg Ehrnrooth
Foundation. B. E. S. was the recipient of studentships from the
MacLaughlin Centre for Molecular Medicine and the CIHR. S. G. holds
the Pitblado Chair in Cell Biology and is cross-appointed to the Department
of Biochemistry of the University of Toronto.

Lysosomal pH measurements
pH measurements were performed by ratiometric imaging of pH-sensitive
dextrans targeted to lysosomes using a pulse-chase protocol. RAW264.7
cells were incubated in serum-free growth medium for 30 min and then
labeled with a mixture of 0.05 mg/mL OG488-dextran and 0.25 mg/mL
FITC-dextran for 620 h at 37 C. This combination of dyes with disparate
pKa values was selected to cover a wide range of pH. Cells were subsequently washed, chased under serum-free conditions for 30 min to 1 h and
imaged with the microscopy system described for FRET, using alternate
excitation through 485 10-nm and 438 12-nm filters, a 505-nm dichroic
mirror and a 535 20-nm emission filter. At the end of each experiment,
an in situ calibration was performed by bathing the cells in K+ -rich solution
containing 20 mM HEPES, 2-(N -morpholino)ethanesulfonic acid (MES) or
acetate, as appropriate, plus 10 g/mL nigericin and buffered to a pH range
of 47, as described (16).

Labeling of cells with other fluorescent markers

To label lysosomes with Alexa 488-dextran or TMR-dextran for localization,
RAW264.7 cells were incubated in serum-free growth medium for 30 min
and then labeled with 0.10.25 mg/mL dextran for 20 h at 37 C. After
extensive washing, cells were labeled with Rh-PE or DACCA-PE and
chased as described above. To label lysosomes with green DQ-BSA, cells
were incubated with 50 g/mL DQ-BSA for 1 h at 37 C before labeling
with the fluorescent lipids. To label mitochondria, RAW264.7 cells were
treated with 500 nM Mitotracker green.

Spinning disc confocal fluorescence microscopy

To assess the detailed localization of Rh-PE and DACCA-PE in the cells
relative to organellar markers, images were acquired using a Quorum
WaveX (Guelph) spinning disc confocal microscopy system based on a
Zeiss Axiovert 200M microscope. Cells were maintained at 37 C using

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Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Figure S1: Fluorescence intensities of Rh-PE and DiBAC4 (3) in
external calibration samples. Rh-PE-labeled RAW264.7 cells were
incubated in K+ -rich buffer containing 1 M gramicidin, 1 M CcA
and the indicated concentrations of DiBAC4 (3). Background-subtracted
fluorescence intensities of Rh-PE and DIBAC4 (3) in cells from 13 to 19
imaged fields from a representative experiment are shown. Data are
means SD.
Figure S2: Comparison of FRET imaging using DiBAC4(3) as the donor
and either fluid-phase rhodamine B-dextran or membrane-bound
Rh-PE as the acceptor. The images were acquired the same day in
parallel experiments using the same cell culture and the same acquisition
settings for each individual channel to make the intensity measurements
comparable. Scale bar = 10 m.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.

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