A lysosomal chloride ion-selective fluorescent probe for biological applications

Article abstract of DOI:10.1039/C8SC04084B

Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions, measurements of their levels are of great importan

Full text of DOI:10.1039/C8SC04084B

Volume 10 Number 1 7 January 2019 Pages 1–334
Chemical
Science
rsc.li/chemical-science
ISSN 2041-6539
EDGE ARTICLE
Injae Shin et al.
A lysosomal chloride ion-selective fluorescent probe for
biological applications

Chemical
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EDGE ARTICLE
A lysosomal chloride ion-selective fluorescent
probe for biological applications†
Cite this: Chem. Sci., 2019, 10, 56
All publication charges for this article Sang-Hyun Park,‡ Ji Young Hyun‡ and Injae Shin
*
have been paid for by the Royal Society
of Chemistry
Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride
channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions,
measurements of their levels are of great importance to understand lysosome-associated biological
ꢀ
events. However, appropriate probes to selectively detect Cl ions within acidic lysosomes have not
ꢀ
been developed to date. In this study, we prepared MQAE-MP, a lysosomal Cl -selective fluorescent
probe, and applied it to gain information about biological processes associated with lysosomes. The
ꢀ
fluorescence of MQAE-MP is pH-insensitive over physiological pH ranges and is quenched by Cl with
ꢀ1
ꢀ
a Stern–Volmer constant of 204 M . Because MQAE-MP detects lysosomal Cl selectively, it was
ꢀ
employed to assess the effects of eleven substances on lysosomal Cl concentrations. The results show
ꢀ
that lysosomal Cl concentrations decrease in cells treated with substances that inhibit proteins
responsible for lysosomal membrane stabilization, induce lysosomal membrane permeabilization, and
ꢀ
transport lysosomal Cl to the cytosol. In addition, we investigated the effect of lysosomal chloride ions
on the fusion of autophagosomes with lysosomes to generate autolysosomes during autophagy
Received 13th September 2018
Accepted 8th November 2018
ꢀ
inhibition promoted by substances. It was found that changes in lysosomal Cl concentrations did not
2+
affect the fusion of autophagosomes with lysosomes but an increase in the cytosolic Ca concentration
DOI: 10.1039/c8sc04084b
blocked the fusion process. We demonstrate from the current study that MQAE-MP has great potential
ꢀ
rsc.li/chemical-science
as a lysosomal Cl -selective fluorescent probe for studies of biological events associated with lysosomes.
ꢀ
suggest that the Cl inux into lysosomes mediated by CLC-7 (a
Introduction
ꢀ
+
Cl /H antiporter) is the principal process occurring during
5
ꢀ
A lysosome is an intracellular organelle that plays a key role in lysosome acidication. As a consequence, the Cl concentra-
degrading and recycling intracellular biomolecules and extra- tion (more than 80 mM) in the lysosome is higher than that in
cellular materials delivered via endocytosis and phagocytosis by the cytosol (5–20 mM) in order to alleviate the charge imbalance
the action of lysosomal hydrolases. This subcellular compart- resulting from the maintenance of the low lysosomal pH.
ment is crucial for autophagy (or a self-eating process) which is Owing to the biological signicance of lysosomal Cl , the
1
6
ꢀ
involved in the maintenance of cellular homeostasis and cell measurement of its level is key to understanding lysosome-
2
survival under the conditions of nutrient deciency. During associated biological events. However, uorescent probes to
ꢀ
autophagy, cytoplasmic constituents are sequestered in auto- selectively detect Cl within acidic lysosomes have not been
phagosomes and subsequently delivered to lysosomes for developed thus far. Consequently, it is in great demand to
digestion by lysosomal hydrolases to produce recyclable prod- create uorescent probes that are pH-insensitive over a broad
ꢀ
ucts. Lysosomal enzymes normally are stable and active in the pH range and selectively monitor Cl within lysosomes.
3
acidic pH range. The low pH (4.5–5.0) inside lysosomes is
To date, engineered uorescent protein-based probes have
+
achieved by the action of vacuolar H -ATPase (V-ATPase), which been constructed and utilized to detect intracellular chloride
pumps protons into the lysosome lumen by using energy ions. However, these probes are not applicable to monitoring
generated by ATP hydrolysis. To maintain electroneutrality lysosomal Cl owing to their pH-sensitivity.
7
ꢀ
7,8
4
On the
during proton pumping, anions must enter lysosomes or other hand, a few chemical uorescent probes have been
9
–12
cations must exit from lysosomes. Multiple previous studies exploited to detect intracellular chloride ions.
For example,
bromide
1
(
-(ethoxycarbonylmethyl)-6-methoxyquinolinium
MQAE) is a pH-insensitive, Cl -selective uorescent probe that
has been employed to detect cytosolic chloride ions (Fig. 1a).
ꢀ
9
Center for Biofunctional Molecules, Department of Chemistry, Yonsei University, Seoul
0
†
1
‡
3722, Republic of Korea. E-mail: injae@yonsei.ac.kr
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8sc04084b
This probe has a relatively large Stern–Volmer constant (200
ꢀ1
ꢀ
M
) for uorescence quenching by Cl . A bisacridinium-based

uorescent probe, with a relatively small Stern–Volmer constant
S.-H. P. and J. Y. H. contributed equally.
56 | Chem. Sci., 2019, 10, 56–66
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ꢀ
ꢀ
Fig. 1 Effect of pH on the fluorescence emission of the fluorescent Cl probes. (a) Chemical structures of the fluorescent Cl probes. (b)
Fluorescence spectra of each probe (100 mM) in 50 mM sodium phosphate buffer at pH 3.0–8.0 were recorded on a fluorimeter with excitation at
3
50 nm wavelength (FI ¼ fluorescence intensity). (c) Stern–Volmer constants for quenching of the fluorescence of MQAE and MQAE-MP by Clꢀ
at various pHs.
ꢀ
1
ꢀ
(
36 M ) for Cl quenching, was observed to be a pH- performed owing to the absence of suitable uorescent probes.
ꢀ
insensitive, Cl -selective probe applicable to measuring endo- In the study described below, we have remedied this deciency
somal chloride ions. Recently, a ratiometric uorescent probe, by developing a uorescent probe, MQAE-MP, which selectively
-methoxyquinolinium-dansyl (MQ-DS), was developed to detects Cl in lysosomes. Our ndings show that MQAE-MP
detect intracellular Cl but is inappropriate to selectively uorescence is pH-insensitive in the physiological pH range
10
ꢀ
6
ꢀ
ꢀ
11
ꢀ
monitor lysosomal Cl . Thus, no chemical probes have been and that its uorescence is efficiently quenched by Cl with
ꢀ1
devised for selectively determining the levels of lysosomal a Stern–Volmer constant of 204 M without interference from
chloride ions. cations and non-halide anions. Moreover, MQAE-MP was
Despite the biological signicance of lysosomal chloride successfully utilized to determine the effects of various
ꢀ
ꢀ
ions, research focusing on lysosomal Cl has not been actively substances on lysosomal Cl concentrations. We found that
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ꢀ
ꢀ
lysosomal Cl concentrations were reduced in cells treated with procedure shown in Scheme 1a. The analysis of the Cl
substances that inhibit proteins responsible for lysosomal concentration-dependent response of MQAE-MP-1 at pH 3–8
ꢀ
membrane stabilization, promote lysosomal membrane per- showed that its uorescence was gradually quenched as the Cl
ꢀ
meabilization (LMP), and transport lysosomal Cl to the concentration increased (Fig. S1†) but was highly pH-sensitive
cytosol. Based on these results, we also investigated whether unlike that of MQAE (Fig. 1b and S1, S2†).
lysosomal chloride ions have an inuence on the fusion of
To circumvent this problem, MQAE-MP-2 (Fig. 1a) contain-
autophagosomes with lysosomes to generate autolysosomes ing a relatively long oligoethylene glycol tether between the
during the inhibition of autophagy induced by substances. The amide NH and morpholine groups was prepared by using the
ꢀ
results showed that alterations in lysosomal Cl concentrations procedure shown in Scheme 1b. The analysis of the pH-
ꢀ
do not affect the fusion of autophagosomes with lysosomes but dependent response of the uorescence of MQAE-MP-2 to Cl
2
+
the fusion process was blocked when cytosolic Ca concentra- showed that its emission intensity was reduced in a concentra-
tions are increased. The combined results demonstrate that tion-dependent manner by Cl (Fig. 1b and S3†). However,
MQAE-MP has great potential for use as a lysosomal Cl -selec- although to a lesser extent than that of MQAE-MP-1, the uo-
ꢀ
ꢀ
tive uorescent probe for studies of biological events associated rescence of MQAE-MP-2 was still sensitive to pH. We assumed
with lysosomes.
from this study that the morpholine moiety in MQAE-MP-1 and
MQAE-MP-2 might affect the uorescence of the 6-methox-
yquinolinium group depending on pH. To test this proposal,
the uorescence of MQAE was measured in the presence of
various concentrations of N-methylmorpholine (NMM) in a pH
range of 3.0–8.0. The results showed that as the pH of solutions
Results
ꢀ
Design, synthesis and characterization of Cl -sensitive

uorescent probes
ꢀ
9
The known Cl -sensitive uorescent probe MQAE was used as containing MQAE and NMM increased from 3.0 to 8.0, NMM
ꢀ
the platform to design lysosome-targeting, Cl -sensitive uo- affected the uorescence of 6-methoxyquinolinium to a greater
rescent probes. To enable lysosome targeting, the ethyl ester extent presumably through the interaction of free NMM with 6-
moiety in MQAE was changed to an amide group containing methoxyquinolinium (Fig. S4†).
13
a morpholine moiety which is a lysosome-targeting motif.
On the basis of these results, we separated morpholine and
MQAE-MP-1 (Fig. 1a), the rst probe constructed based on this quinolinium groups by using a rigid rather than exible tether
design that possessed an ethylene tether between the amide NH to alleviate or eliminate the effect of a morpholine moiety on the
and morpholine groups, was synthesized by using the uorescence of a quinolinium group. MQAE-MP (Fig. 1a), which
ꢀ
Scheme 1 Synthesis of lysosome-targeting fluorescent Cl probes: (a) MQAE-MP-1, (b) MQAE-MP-2 and (c) MQAE-MP.
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contains a rigid isonipecotic acid moiety as the tether between were incubated with MQAE for comparison purposes. The
amide NH and morpholine groups, was prepared by using the analysis of confocal uorescence microscopy images of cells
procedure shown in Scheme 1c. To our pleasure, the analysis of showed that uorescence arising from MQAE was distributed
the emission properties of MQAE-MP demonstrated that its throughout the cytoplasm (Fig. 3). Importantly, the uorescence

uorescence was insensitive to pH over the 3.0–8.0 range and of MQAE-MP overlapped well with that of LysoTracker Red
ꢀ
ꢀ
was quenched in a Cl concentration-dependent manner (Fig. 3). Because the lysosomal Cl concentration is normally
ꢀ
(
Fig. 1b and S5a†). MQAE-MP exhibited two absorption bands higher (ꢁ80 mM) than the cytosolic Cl concentration (5–20
14
with maximum wavelengths at 320 nm and 350 nm (Fig. S6†) mM), the uorescence intensity of MQAE-MP in lysosomes
and an emission band with a maximum wavelength at 460 nm was observed to be weaker than that of MQAE. However, cells
ꢀ
(
Fig. 1b), which were quite close to those of MQAE (Fig. 1b and cultured in Cl -decient buffer exhibited stronger uorescence
S6†). In addition, emission from MQAE-MP was quenched by of MQAE-MP than cells in normal media, in accordance with the
ꢀ
ꢀ
Cl in the range of 0–250 mM with a Stern–Volmer quenching fact that the lysosomal Cl concentration is reduced when cells
ꢀ
ꢀ1
ꢀ
15
constant (Cl quenching sensitivity) of 204 M at pH 7.0, are cultured in Cl -decient buffer. It should be noted that
a value that is close to that of MQAE (202 M ) (Fig. 1c and S2b, because lysosomal Cl concentrations are normally reduced in
S5b†). The uorescence response of MQAE-MP to various cells treated with substances that disrupt the function of lyso-
anions and cations was assessed. As shown in Fig. 2 and S7,† somes, changes in the levels of lysosomal Cl could be readily
ꢀ1
ꢀ
9
ꢀ
+
+
emission from MQAE-MP was not affected by cations (Na , K , determined by using MQAE-MP (vide infra). Overall, the ndings
2
+
2+
+
+
ꢀ
Ca , Mg , NH and CH NH ), F , and non-halide anions indicate that MQAE-MP accumulates mainly in lysosomes
4
3
3
(
bicarbonate, sulfate, phosphate and nitrate) like that of MQAE. owing to the lysosome-targeting morpholine group and that it
ꢀ
In addition, these ions did not interfere with the uorescence selectively and efficiently detects lysosomal Cl .
response of MQAE-MP to Cl (Fig. 2b).
ꢀ
Also, a study was conducted to elucidate the time-
ꢀ
dependence of detection of lysosomal Cl using MQAE-MP.
ꢀ
For this purpose, HeLa cells cultured in Cl -decient buffer
ꢀ
Detection of lysosomal Cl ions in cells using MQAE-MP
were treated with MQAE-MP while confocal uorescence
ꢀ
The desirable properties (pH-insensitive and Cl -sensitive) of microscopy images of cells were collected during a 2 h time
MQAE-MP in aqueous solutions stimulated a study probing its period. The results showed that the uorescence intensity of
ꢀ
ability to detect Cl in lysosomes of cells. For this study, HeLa MQAE-MP in cells rapidly increased and nearly reached satu-
ꢀ
cells cultured in normal media or Cl -decient buffer were ration aer ca. 30 min incubation (Fig. S8†). The combined
treated with MQAE-MP and LysoTracker Red. Also, the cells ndings indicate that MQAE-MP is applicable for monitoring
ꢀ
Cl inside acidic lysosomes.
ꢀ
Analysis of the effects of substances on lysosomal Cl
concentrations using MQAE-MP
Next, MQAE-MP was employed to determine changes in lyso-
ꢀ
somal Cl concentrations promoted by eleven substances that
affect the function of lysosomes (Fig. 4). The substances used
for this study include synthetic ion transporters (diamide-
strapped calix[4]pyrrole-1 (DSC4P-1) and squaramide-3 (SA-3)),
inhibitors of a proton pump V-ATPase (balomycin A1 (BfA1))
and a chloride channel blocker (niumic acid (NFA)), lysoso-
motropic amines (ammonium ions, methylammonium ions,
hydroxychloroquine (HCQ) and polyethylenimine (PEI)), and
inhibitors of lysosomal proteins (chlorpromazine (CPZ), apop-
tozole (Az) and leupeptin (LP)). HeLa cells were incubated for
6
h with several concentrations of each substance in normal
culture media and then treated for 30 min with non-cytotoxic
MQAE-MP (Fig. S9†) and acridine orange, which is used to
16
detect acidic vesicles such as lysosomes. Cell images were
obtained by using confocal uorescence microscopy.
The effects of two synthetic ion transporters, DSC4P-1 and
ꢀ
Fig. 2 Fluorescence response of (a) MQAE (100 mM) and (b) MQAE-MP SA-3, on lysosomal Cl concentrations and pH were assessed
(
100 mM) to indicated ions (50 mM) in buffer (lex ¼ 350 nm). Gray bars

rst. Our previous studies showed that DSC4P-1 and SA-3
represent F /F ratios after addition of various competing ions to
0
ꢀ
promoted increases in the cytosolic Cl concentration by their
ability to transport extracellular Cl with higher concentrations
a solution of each probe (mean ꢂ s.d., n ¼ 3). Dark gray bars represent
ꢀ
ꢀ
F
0
/F ratios after addition of Cl (50 mM) to a solution of each probe in
ꢀ
(120 mM) to the cytosol with lower Cl concentrations (5–20
0
the presence of competing ions (F : fluorescence of buffer only; F:
fluorescence of buffer containing Cl ).
ꢀ
17,18
mM).
However, the determination of changes in lysosomal
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ꢀ
ꢀ
Fig. 3 Detection of Cl ions in cells using MQAE-MP or MQAE. HeLa cells were incubated in (a) normal media and (b) Cl -deficient buffer for 6 h
and then treated with 100 nM LysoTracker Red and (left) 5 mM MQAE-MP or (right) 1 mM MQAE for 30 min. Cell images were obtained by using
confocal fluorescence microscopy (scale bar: 10 mm). Fluorescence intensities of MQAE-MP, MQAE and LysoTracker Red in the cells were
analyzed by using ZEN software. Graph shows the fluorescence intensity of (left) MQAE-MP (cyan) and LysoTracker (red) or (right) MQAE (cyan)
and LysoTracker (red) at positions along the line across cells. Experiments were repeated three times, giving similar results.
ꢀ
Cl concentrations caused by these transporters was not con- a higher concentration (more than 80 mM) to the cytosol with
ꢀ
6,14
ducted at that time owing to the lack of a suitable lysosomal Cl

a lower concentration (5–20 mM).
However, it could be
ꢀ
uorescent probe. The results of the current effort using MQAE- possible that the decrease in the lysosomal Cl concentration is
MP clearly indicate that DSC4P-1 does not affect the lysosomal caused by its ready exit across lysosomal membranes disrupted
ꢀ
Cl concentration and pH (Fig. 5a and S10†). The ndings by SA-3. To determine whether SA-3 induces LMP, the release of
suggest that this transporter does not have the ability to trans- lysosomal cathepsin B into the cytosol of treated cells, which is
ꢀ
19
port lysosomal Cl to the cytosol.
evidence for LMP, was examined. HeLa cells were incubated
In marked contrast to DSC4P-1, SA-3 promoted a dose- for 6 h with SA-3, along with DSC4P-1, and the cathepsin B
ꢀ
dependent decrease in the level of lysosomal Cl , as judged activity of the isolated cytosolic fraction of the treated HeLa cells
from an increase in the intensity of uorescence from MQAE- was then measured by using a uorogenic substrate, MR-
17
MP, and increased the lysosomal pH, based on the observa- (RR)2. The results showed that cathepsin B activity was not
tion of the disappearance of red uorescence from acridine detected in the cytosol of the cells treated with SA-3,
1
5
ꢀ
orange (Fig. 5a and S10†). The reduced level of lysosomal Cl
a phenomenon also seen in cells treated with DSC4P-1
17
caused by SA-3 could be a result of the previous suggestion
(Fig. 5b). The ndings indicate that SA-3 does not induce LMP
ꢀ
ꢀ
that SA-3 has the ability to transport Cl from lysosomes with and, consequently, promotes a decrease in the lysosomal Cl
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Fig. 4 Chemical structures of the substances used in this study (ASM: acid sphingomyelinase).
ꢀ
concentration through its lysosomal Cl transport activity. proton leakage under conditions where pumping of protons
Concomitantly, SA-3 increased the lysosomal pH as a result of into lysosomes is suppressed by either BfA1 or niumic acid
20,21
5
proton leakage which takes place in lysosomes.
Together (Fig. S11b and c†).
with the previous results, the ndings indicate that while
The effects of lysosomotropic amines (ammonium ions,
ꢀ
DSC4P-1 transports extracellular Cl to the cytosol without methylammonium ions, hydroxychloroquine and poly-
ꢀ
ꢀ
transporting lysosomal Cl to the cytosol, SA-3 has the ability to ethylenimine (PEI)) on lysosomal Cl levels and pH were
ꢀ
ꢀ
25
transport extracellular Cl as well as lysosomal Cl to the assessed. It is known that uncharged forms of lysosomotropic
cytosol. amines freely diffuse across membranes and their protonated
Next, confocal uorescence microscopy images of cells (non-diffusible) forms accumulate in acidic intracellular
26
treated with BfA1 and niumic acid were analyzed to determine compartments such as lysosomes. When trapped in lyso-
ꢀ
their effects on lysosomal Cl concentrations and pH. BfA1 is an somes, lysosomotropic amines induce lysosomal osmotic
inhibitor of the proton pump V-ATPase and thus causes an stress, leading to the disruption of lysosomal membranes and
2
2,23
25,27–29
increase in lysosomal pH.
Niumic acid is a cellular chloride induction of LMP.
Cell image analyses revealed that each
ꢀ
24
channel inhibitor that regulates the Cl concentration in cells.
However, the effects of these substances on lysosomal Cl
of the tested lysosomotropic amines caused a dose-dependent
decrease in lysosomal Cl concentrations and an increase in
ꢀ
ꢀ
concentrations have not been determined. The results of cell lysosomal pH (Fig. 5a and S10†). As expected, lysosomotropic
image analyses showed that BfA1 and niumic acid did not alter amines caused LMP, as judged by observations that cathepsin B
ꢀ
the levels of lysosomal Cl but they did induce an increase in activities were detected in the isolated cytosol of the treated
the lysosomal pH in a dose-dependent manner (Fig. 5a and cells (Fig. 5b). However, when leupeptin, an inhibitor of
30
S10†). To determine whether BfA1 and niumic acid promote cathepsin B, was present in the isolated cytosol of the treated
LMP, cathepsin B activities in the cytosol of HeLa cells treated cells, cathepsin B activities were not detected. On the basis of
+
ꢀ
with these substances were determined. In each case, cathepsin these observations, we conclude that lysosomal H and Cl exit
B activity was not detected in the cytosol of treated cells from lysosomes to the cytosol across disrupted lysosomal
(Fig. 5b), indicating that both BfA1 and niumic acid do not membranes of the cells treated with lysosomotropic amines.
induce LMP. Collectively, the ndings suggest that either BfA1
or niumic acid blocks Cl entry into lysosomes because V- (chlorpromazine for acid sphingomyelinase (ASM), apoptozole
The effects of the three inhibitors of lysosomal proteins
ꢀ
ATPase and CLC-7 cooperatively function, thus leading to no for lysosomal Hsp70, and leupeptin for cathepsin B) on lyso-
ꢀ
4
ꢀ
changes in lysosomal Cl concentrations (Fig. S11b and c†).
somal Cl concentrations and pH were determined. Chlor-
Moreover, the lysosomal pH increased in cells treated with promazine is an inhibitor of ASM responsible for the cleavage of
either BfA1 or niumic acid is likely to be a consequence of sphingomyelin into ceramide and phosphorylcholine that are
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Fig. 5 Detection of lysosomal chloride ions in cells treated with various substances using MQAE-MP. (a) HeLa cells were incubated for 6 h with
+
+
m
ꢀ1
4 3 3
each substance (concentration: 40 mM DSC4P-1, 5 mM SA-3, 5 nM BfA1, 5 mM NFA, 30 mM NH , 30 mM CH NH , 50 mM HCQ, 5 g mL PEI, 20
mM CPZ, 5 mM Az and 50 mM LP) and then treated for 30 min with 5 mM MQAE-MP or 100 nM acridine orange (AO). Cell images were obtained by
using confocal fluorescence microscopy (scale bar: 10 mm). Graph shows the fluorescence intensities (FI) of cells treated with indicated
concentrations of each substance for 6 h (see Fig. S10† for complete images). Fluorescence intensities of MQAE-MP in the cells were analyzed by
using ZEN software. (b) HeLa cells were treated with each substance for 6 h. Cathepsin B activity of the isolated cytosolic fraction was measured
by using a fluorogenic substrate MR-(RR)
2
in the absence and presence of leupeptin (mean ꢂ s.d., n ¼ 3).
3
1,32
involved in the stabilization of lysosomal membranes.
Apoptozole is an inhibitor of lysosomal Hsp70,
times with SA-3, four lysosomotropic amines, chlorpromazine
which acts as and apoptozole, all of which affect the levels of lysosomal
a lysosomal membrane stabilizer through its interaction with chloride ions. The results showed that whereas SA-3 promoted
33,34
35
ꢀ
lysosomal membranes. Leupeptin is a naturally occurring a relatively slow decrease in the lysosomal Cl concentration,
inhibitor of cathepsin B, which degrades proteins inside lyso- lysosomotropic amines, chlorpromazine and apoptozole
somes. As shown in Fig. 5a and S10,† the treatment of cells caused relatively rapid decreases in lysosomal Cl concen-
30
ꢀ
with chlorpromazine led to a reduction in the level of lysosomal trations (Fig.
6
and S12†). The ndings suggest that
ꢀ
ꢀ
Cl and an increase in lysosomal pH. In addition, chlorprom- substances with the ability to induce LMP accelerate Cl efflux
36
azine induced LMP, as judged by the observation that from lysosomes into the cytosol more rapidly than the
cathepsin B was released from lysosomes to the cytosol of the synthetic ion transporter SA-3 which does not induce LMP. It
+
ꢀ
treated cells (Fig. 5b). Consequently, lysosomal H and Cl ions is worth mentioning that although LMP-inducing agents
exited across disrupted membranes of lysosomes of the cells promote the release of cathepsin B into the cytosol, the uo-
treated with chlorpromazine. Similarly, apoptozole caused rescence arising from Lysotracker and MQAE-MP is detectable
ꢀ
37,38
a decrease in the level of lysosomal Cl ions and an increase in in lysosomes.
lysosomal pH (Fig. 5a and S10†) as a result of the ability of
Collectively, the results of the study aimed at determining
ꢀ
apoptozole to destabilize lysosomal membranes and the the effects of substances on lysosomal Cl concentrations using
34
consequent induction of LMP (Fig. 5b). In contrast to chlor- MQAE-MP show that (1) the synthetic ion transporter, SA-3,
ꢀ
+
promazine and apoptozole, leupeptin did not cause any change promotes decreases in lysosomal Cl and H concentrations
ꢀ
ꢀ
+
in the lysosomal Cl concentration and pH (Fig. 5a and S10†). but DSC4P-1 does not affect lysosomal Cl and H concentra-
ꢀ
Time-dependent changes in the levels of lysosomal Cl
tions, (2) inhibitors of a proton pump V-ATPase (BfA1) and
+
promoted by these substances were also investigated using a chloride channel (NFA) cause decreases in H concentrations
MQAE-MP as the probe. HeLa cells were incubated for various without affecting Cl concentrations, and (3) substances that
ꢀ
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in the cells treated with BfA1 which does not affect the lyso-
ꢀ
somal Cl concentration (Fig. 7a). The nding indicates that
BfA1 blocks the fusion of autophagosomes with lysosomes,
23
which is in agreement with the recent nding. As expected, the
2
+
cytosolic Ca concentration greatly increased (ꢁ2 times) in the
23
cells treated with BfA1, as judged from the observation of an
2
+
increase in the uorescent intensity of Ca -sensitive probe
17
Fluoro-4NW (Fig. 7b). In contrast to BfA1, the cells treated with
ꢀ
niumic acid which does not affect the lysosomal Cl concen-
tration exhibited signicant colocalization of LAMP2A and p62
(
Fig. 7a), indicating that niumic acid allows the fusion of
2
+
lysosomes with autophagosomes. In addition, the cytosolic Ca
concentration was found to be unaltered in the cells treated
with niumic acid (Fig. 7b). Consequently, the results show that
ꢀ
the two substances which do not change lysosomal Cl
Fig. 6 Time-dependent detection of lysosomal chloride ions in cells concentrations differently affect the fusion of autophagosomes
ꢀ
treated with various substances using MQAE-MP. HeLa cells were
with lysosomes, suggesting that lysosomal Cl may have no
inuence on the fusion process.
incubated with each compound (concentration: 5 mM SA-3, 30 mM
+
+
ꢀ1
NH
mM Az) for indicated times and then treated with 5 mM MQAE-MP for
0 min (mean ꢂ s.d., n ¼ 3). Graphs show the fluorescence intensities
4 3 3
, 30 mM CH NH , 50 mM HCQ, 5 mg mL PEI, 20 mM CPZ and 5
Cell image analysis also revealed that the fusion of auto-
phagosomes with lysosomes took place in cells treated with
3
(
FI) of cells treated with each substance for indicated times (see LMP-inducing substances, such as ammonium ions, chlor-
Fig. S12† for cell images).
promazine and apoptozole (Fig. 7a). In the cells treated with
2
+
each of LMP-inducing substances, cytosolic Ca concentrations
were not changed (Fig. 7b). These phenomena were also
observed in cells incubated with leupeptin (Fig. 7b). As
a consequence, the LMP-inducing substances, which caused
ꢀ
+
induce LMP cause decreases in lysosomal Cl and H
concentrations.
ꢀ
decreases in lysosomal Cl concentrations, as well as leupeptin,
which did not affect the lysosomal Cl concentration, allow the
fusion of autophagosomes with lysosomes. The ndings also
support the conclusion that the fusion process may not be
ꢀ
ꢀ
Effect of lysosomal Cl on the fusion of autophagosomes with
lysosomes
ꢀ
The lysosome is a crucial intracellular compartment for auto- affected by lysosomal Cl .
phagy during which autophagosomes containing cytoplasmic
Interestingly, cells treated with SA-3 with lysosomal chloride
constituents are fused with lysosomes to generate autolyso- transport activity exhibited partially fused vesicles (or puncta)
39
2+
somes. In autolysosomes, engulfed cytoplasmic components even aer 6 h incubation (Fig. 7a). The cytosolic Ca concen-
are degraded by lysosomal enzymes, which normally are stable tration in the cells treated with SA-3 was measured to increase
40
and active at an acidic pH. Recently, it was shown that the by half of that caused by BfA1 (Fig. 7b). Recently, we have found
4
3
fusion of autophagosomes with lysosomes is suppressed at high that SA-3 activates the inositol trisphosphate receptor (IP
3
R)
2
+
23
2+
cytosolic Ca concentrations. In light of this, we wondered responsible for Ca efflux from the ER to the cytosol and,
whether lysosomal chloride ions also have an inuence on the consequently, causes an increase in the cytosolic Ca concen-
fusion process to generate autolysosomes. tration. On this basis, we examined whether an increase in the
2
+
ꢀ
2+
Thus, we assessed the effect of lysosomal Cl on the fusion of cytosolic Ca concentration in the cells treated with SA-3
autophagosomes with lysosomes in cells undergoing autophagy affected the fusion of autophagosomes with lysosomes. For
+
inhibition promoted by SA-3, BfA1, niumic acid, NH
4
, chlor- this purpose, cells were co-treated with SA-3 and an IP
3
R
44
promazine, apoptozole and leupeptin (see Fig. S13† for the inhibitor, 2-aminoethoxydiphenyl borate (2-APB). When 2-APB
inhibition of autophagy by these substances). To accomplish this, was present in the SA-3 treated cells, the cytosolic Ca
2
+
HeLa cells were individually treated for 3 or 6 h with each concentration was reduced to a normal level and the fusion of
substance. The treated cells were immunostained with LAMP2A autophagosomes with lysosomes was comparable to that in the
and p62 antibodies serving as respective lysosome and auto- cells treated with niumic acid, LMP-inducing substances or
41,42
2+
phagosome markers.
If lysosomes and autophagosomes are leupeptin (Fig. 7). Because the cytosolic Ca concentration in
fused in the treated cells, yellow uorescence arising from the cells treated with SA-3 increased to a lesser extent than that
colocalization of lysosome (LAMP2A, green) and autophagosome in the cells treated with BfA1, the fusion of autophagosomes
(
p62, red) markers will be observed. However, discrete uores- with lysosomes was not completely suppressed in the cells
cence arising from LAMP2A and p62 without detecting yellow treated with SA-3 but partially fused vesicles were observed.

uorescence will be seen in the cells treated with substances that Taken together, the ndings suggest that changes in lysosomal
ꢀ
suppress the fusion of autophagosomes with lysosomes.
Cl concentrations have no inuence on the fusion process but
2
+
The analysis of confocal uorescence microscopy images of high cytosolic Ca concentrations are critical to block the
the cells showed that discrete LAMP2A and p62 puncta existed fusion process.
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Fig. 7 Effect of substances on the fusion of autophagosomes with lysosomes. (a) HeLa cells were incubated for 3 or 6 h with an individual
+
4
compound (200 nM BfA1, 5 mM NFA, 5 mM SA-3, 30 mM NH , 20 mM CPZ, 5 mM Az, 50 mM LP, and 5 mM SA-3 + 10 mM 2-APB). The cells were
immunostained with LAMP2A and p62 antibodies. Merged images of the cells immunostained with LAMP2A and p62 antibodies are shown (scale
bar: 10 mm) and white arrows indicate the magnified area. (b) HeLa cells were incubated with 10 mM Fluoro-4NW for 1 h and then treated for 12 h
+
with an individual compound (200 nM BfA1, 5 mM NFA, 5 mM SA-3, 30 mM NH
4
, 20 mM CPZ, 5 mM Az, 50 mM LP, and 5 mM SA-3 + 10 mM 2-APB).
Fluorescence intensities of the treated cells were measured by using a microplate reader (mean ꢂ s.d., n ¼ 3).
ꢀ
membrane stabilization, cause changes in both lysosomal Cl
Conclusions
concentration and pH by inducing LMP. Similar results also
ꢀ
We developed the rst lysosomal Cl -selective uorescent probe arise from studies of cells treated with lysosomotropic amines,
by linking a quinolinium uorophore to a lysosome-targeting which promote the induction of LMP. However, inhibitors of V-
morpholine group. It was found that the nature of the tether ATPase (BfA1) and a chloride channel (niumic acid) increase
ꢀ
between the quinolinium uorophore and morpholine moiety lysosomal pH without affecting lysosomal Cl concentrations.
was key to generating the desired uorescence properties. In contrast, an inhibitor of cathepsin B (leupeptin) does not
ꢀ
Specically, the uorescence of probes, MQAE-MP-1 and MQAE- change either lysosomal Cl concentration or pH. Collectively,
ꢀ
MP-2, containing exible tethers is sensitive to pH, whereas the the ndings indicate that lysosomal Cl concentrations
probe MQAE-MP containing a rigid tether is pH-insensitive. decrease in cells treated with substances that inhibit proteins
ꢀ
Moreover, the uorescence of MQAE-MP is quenched by Cl
responsible for lysosomal membrane stabilization, induce LMP,
ꢀ
1
ꢀ
with a Stern–Volmer constant of 204 M but is not responsive and transport lysosomal Cl to the cytosol.
to cations, F and non-halide anions. Because MQAE-MP
ꢀ
ꢀ
We also examined whether lysosomal Cl concentrations
accumulates mainly in lysosomes and successfully responds affected the fusion of autophagosomes with lysosomes to form
ꢀ
to lysosomal Cl by undergoing a decrease in uorescence autolysosomes. Our results showed that BfA1, which does not
ꢀ
intensity, it was applied to assess the effects of eleven affect the lysosomal Cl concentration but increases the
ꢀ
2+
substances on lysosomal Cl concentrations. Among the cytosolic Ca concentration, suppressed the fusion of auto-
substances, a DSC4P-1 based synthetic ion transporter did not phagosomes with lysosomes. However, ammonium ions,
ꢀ
change both lysosomal Cl concentration and pH, but a SA-3 chlorpromazine and apoptozole, which cause decreases in
ꢀ
2+
based synthetic ion transporter altered both. This observation lysosomal Cl concentrations but do not change cytosolic Ca
might result from the difference in the ability of these concentrations, allowed the fusion of autophagosomes with
ꢀ
substances to transport lysosomal Cl to the cytosol. In addi- lysosomes. In addition, niumic acid and leupeptin, which
ꢀ
tion, chlorpromazine and apoptozole, which inhibit respective affect neither the lysosomal Cl concentrations nor cytosolic
2
+
ASM and lysosomal Hsp70 responsible for lysosomal Ca
concentrations, also allowed the fusion process.
64 | Chem. Sci., 2019, 10, 56–66
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Interestingly, SA-3, which induces a decrease in the lysosomal Aldrich) was added to the cells and the resulting cells were
ꢀ
Cl concentration, caused the partial fusion of autophago- shaken on a rocker on ice for 12 min. The extraction buffer was
2
+
somes with lysosomes because the cytosolic Ca concentration collected and used as the cytosolic fraction. The cytosolic frac-
in the SA-3 treated cells increases by half of that caused by BfA1. tion was treated with 1 mL MR-(RR)2 (ImmunoCytochemistry
Collectively, the ndings provide evidence to support the notion Technologies) in the absence and presence of 20 mM leupeptin
ꢀ
ꢃ
that alterations in lysosomal Cl concentrations do not affect for an additional 4 h at 37 C. The enzyme-catalyzed release of
the fusion of lysosomes with autophagosomes but increases in the uorescent probe was monitored by using an Innite® 200
2
+
cytosolic Ca concentrations are critical to block the fusion PRO multimode microplate reader (excitation wavelength:
process. It should be noted that the disruption of lysosomal 540 nm; emission wavelength: 600 nm).
membranes and an increase in lysosomal pH are also not crit-
ical for the suppression of fusion of autophagosomes with
Immunocytochemistry
lysosomes. It is clear that the investigation described above has
HeLa cells were incubated for 3 or 6 h with each compound at
demonstrated the usefulness of MQAE-MP in studies of how
ꢀ
the indicated concentrations. The treated cells were xed with
substances affect the levels of lysosomal Cl ions. It is antici-
4% formaldehyde in PBS buffer for 15 min. The cells were
pated that MQAE-MP will serve as a useful uorescent probe to
incubated with mouse LAMP2A monoclonal (1:200, Santa Cruz)
and guinea pig p62 polyclonal (1:200, PROGEN) for 1 h at room
temperature followed by incubation with Alexa-Fluoro 488
conjugated goat anti-mouse IgG (1:200, Invitrogen, Molecular
Probes) and Alexa-Fluoro 555 conjugated goat anti-guinea pig
IgG (1:200, Invitrogen, Molecular Probes) for 1 h at room
temperature. The cells were imaged by using confocal uores-
cence microscopy (Zeiss LSM 800).
explore more lysosome-associated biological systems.
Experimental section
Spectral characterization of uorescent Cl probes
ꢀ
ꢀ
Fluorescence quenching measurements with uorescent Cl
probes were performed by excitation at 350 nm. Microliter
aliquots of NaCl (5 M stock) were added to 3 mL of each uo-
rescent probe (100 mM in 50 mM sodium phosphate buffer) at
various pHs (pH 3–8). Stern–Volmer constants were calculated Detection of calcium ions
ꢀ
from the slope of F /F versus [Cl ] plots, where F is the uo-
0
ꢀ
0
HeLa cells were incubated with 10 mM Fluo-4NW in culture
media for 1 h. Aer washing with PBS to remove remaining
Fluo-4NW, the cells were incubated with an indicated
compound for 12 h. Fluo-4 uorescence was measured using
a microplate reader (excitation wavelength: 485 nm; emission
ꢀ
rescence of each Cl probe in the absence of Cl and F in the
ꢀ
presence of Cl .
MQAE-MP staining
HeLa cells in culture media were incubated with each substance wavelength: 538 nm).
at the indicated concentrations and for the indicated times. The
cells were washed three times with DPBS and then incubated
Conflicts of interest
with 5 mM of MQAE-MP and 100 nM of LysoTracker Red
ꢃ
(
ThermoFisher Scientic) for 30 min at 37 C. The cells were
The authors declare no competing nancial interests.
washed twice with DPBS and imaged by using confocal uo-
rescence microscopy (Zeiss LSM 800). The uorescence inten-
sities of uorescent dyes in the cells were quantied using the
mean region of interest (ROI) tool with ZEN soware. Speci-
cally, a constant circular ROI was chosen to encompass the cell
of interest and the same ROI area size was used for background
subtraction.
Acknowledgements
This study was nancially supported by a grant from the
National Creative Research Initiative (2010-0018272) Program
in Korea.
Acridine orange staining
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