Visual monitoring of the lysosomal pH changes during autophagy with a red-emission fluorescent probe

Article abstract of DOI:10.1039/c9tb02551k

Autophagy plays crucial roles in maintaining normal intracellular homeostasis. Molecular probes capable of monitoring lysosomal pH changes during autophagy are still highly required yet challenging to develop. Here, a lysosome-targeting fluorescent pH pro

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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
particles
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Visual monitoring of the lysosomal pH changes during autophagy
with a red-emission fluorescent probe
Xiaodong Wang†a, Li Fan*†a, Yubin Wangb, Caihong Zhangb, Wenting Lianga, Shaomin Shuangb and
Received 00th January 20xx,
Accepted 00th January 20xx
Chuan Dong*a
DOI: 10.1039/x0xx00000x
Autophagy play crucial roles in maintaining normal intracellular homeostasis. Molecular probes capable of monitoring
lysosomal pH changes during autophagy are still highly required yet challenging to develop. Here a lysosome-targeting pH
fluorescent probe RML is presented by introducing a methylcarbitol unit as the lysosome-targeting group to the rhodamine
B, which is highly sensitive to pH changes. RML exhibits remarkable pH-dependent behavior at 583 nm with the fluorescent
enhancement more than 148-fold. The pKa value is determined as 4.96, and the linear response with pH changes from 4.50-
5.70, which is favorable for lysosomal pH imaging. We also confirm that RML diffuses selectively into lysosomes using
confocal fluorescent microscopy. Using RML, we have successfully visualized the autophagy by monitoring the lysosomal
pH changes.
www.rsc.org/
autolysosome, in which the cellular components are degraded by
virtue of acidic hydrolase (pH 4.5-5.5).14,15,36 Owing to inner
micro-environmental difference between the lysosome and
autophagosome, the lysosomal pH changes during the autophagy.
Accordingly, it might be an efficient and particle method for
visualization autophagy by monitoring the lysosomal pH
changes.
Introduction
Autophagy (“self-eating” in Greek) is a lysosomal degradation
process for clearing dysfunctional or damaged organelles and
proteins, as well as providing nutrients for cells.1,2 Most of the
widely used strategies for monitoring autophagy are based on
transmission electron microscopy (TEM), and the visualization
of autophagosome-related proteins by western blotting
(Atg8/LC3) or genetically encoded fluorescent proteins (GFP-
LC3/p62).3-6 However, they might suffer from some inherent
drawbacks. For example, they either require cell fixation (i.e. in
dead cells) in the TEM and western blotting methods7,8, or rely
on complicated design and transfection process in the use of
genetically encoded fluorescent proteins.8-10 Besides, it’s
difficult to achieve rapid and low-cost monitoring of autophagy
in live cells through these strategies.11-13 Thus, the development
of novel method for visualization of autophagy in an efficient
and cost-effective manner is still highly demand.
So far, only a few pH fluorescent probes are developed for
visual monitoring autophagy. Kim’s group designed
mitochondria-targeting pH probe for monitoring of mitochondria
acidification undergoing mitophagic elimination during
autophagy.16 Unfortunately, the probe has ultraviolet excitation
and short emission wavelength (no more than 550 nm), which is
highly absorbed by biomolecules. Then, Iwashita designed a
Mtphagy Dye with emission in the long-wavelength region, for
minimizing photodamage and avoiding the influence of cell
autofluorescence.17 Shangguan’s group reported a cyanine dye
with red emission for simultaneously probe mitochondria and
autolysosomes.13 Although the above probes have been
successfully applied in visualization of autophagy, they were
both mitochondria-targetable pH probes for monitoring the pH
changes of mitochondria-containing autolysosomes during the
mitochondria-associated autophagy.13,16-19 Meanwhile, the
complex synthetic process and easy photobleaching of cyanine
dyes may narrow their practical application. Very recently,
Meng’s group synthesized a benzimidazole-decorated two-
photon fluorescent probe for visualization of starvation-induced
autophagy via detecting pH changes in the lysosomes.11 But the
probe still has fluorescent emission within ultraviolet region.
Another key issue is that the probe as well as most of the
lysosome-targetable pH probes,
In general, autophagy is initiated by the formation of
autophagosomes, which can engulf dysfunctional organelles
(such as mitochondria) or unnecessary cellular components.
Subsequently, the autophagosomes fuse with lysosomes to form
aInstitute of Environmental Science, Shanxi University, Taiyuan, 030006, P. R.
China.
bCollege of Chemistry and Chemical Engineering, Shanxi University, Taiyuan,
030006, P. R. China.
Email: dc@sxu.edu.cn; fanli128@sxu.edu.cn
Fax: +86-531-7011011; Tel: +86-531-7011011
†These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/
x0xx00000x
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Scheme 1 Illustration of visual monitoring of autophagy utilizing the lysosome-targeting pH fluorescent probe RML in cells.
including the commercial LysoSensor and lysoTrackers, has
Results and discussion
weakly basic nitrogen-containing side chains (i. e. morpholine
Optical Properties of RML
or N, N-dimethylethylenediamine) that making them
The optical properties of RML were measured in B-R buffer
solution (40 mM) containing 2.5 % DMSO for UV-vis spectra
and 1 % DMSO for fluorescent spectra, respectively. As
expected, the probe exhibited highly sensitive response to pH.
As shown in Fig. S2 (ESI†), almost no absorption RML was
colourless at pH 7.4. Whereas the pH reducing to 4.50, an
absorbance at 561 nm is observed and increased gradually (ε561
nm = 9.36 × 103 M-1 cm-1), with the solution changing to pink.
Fig. 1a shows the pH-dependent fluorescence spectroscopy of
RML. Similar to the absorption spectra, there is no
fluorescence observed under the near-neutral condition. Upon
the pH decreasing from 7.40 to 4.40, a more than 148-fold
fluorescence enhancement at 583 nm appeared (Fig. 1a).
Meanwhile, the fluorescence colour of the solution turned into
red under the UV lamp of 365 nm (Inset of Fig. 1a). The pKa
value was determined as 4.96, and the linear response with pH
changed from 4.50-5.70, which is favourable for lysosomal pH
imaging. Moreover, the fluorescence quantum yield of RML in
B-R buffer solution increased from 0.012 to 0.214 with the pH
decreasing from 7.40 to 4.40 (using rhodamine B (Φ = 0.69 in
MeOH) as a reference).
selectively locate in acidic lysosomes, and would inevitably
exhibit an “alkalizing effect” on lysosomes.7, 20-28 Therefore, we
aim to design novel pH probes with long-wavelength emission
and negligible “alkalizing effect” on lysosomes for monitoring
of autophagy by detecting the lysosomal pH changes.
RML is designed by introducing a methylcarbitol unit as
the lysosome-targeting group to the rhodamine B (Scheme 1
and Scheme S1, ESI†), which is highly sensitive to pH changes
due to the unique spirocycle group.24,29-34 The spirocyclic ring
usually is closed under neutral pH conditions, making
rhodamine B completely nonfluorescent. Whereas protonation
of N induces spirocyclic ring opening, which leads to
remarkable fluorescence enhancement. Furthermore, other
excellent
photophysical
properties
including
high
photostability and quantum yield, together making rhodamine
B suitable for long-time imaging in living cells. On the other
hand, methylcarbitol is reported as a novel lysosome-targeting
unit for effectively avoiding the “alkalizing effect” on
lysosomes.24 As expected, RML exhibits remarkable pH-
dependent behavior at 583 nm with the fluorescent
enhancement more than 148-fold. The pKa value is determined
as 4.96, and the linear response with pH changes from 4.50-
5.70, which is favorable for lysosomal pH imaging. We also
demonstrated the applicability of RML for visual monitoring
of autophagy by imaging the lysosomal pH changes.
To confirm the fluorescent enhancement is pH-selective,
we further measured the fluorescent intensities of RML
together with cations, anions, or amino acids at pH 7.40 and pH
4.40, respectively. No noticeable fluorescence enhancement
was observed, confirming that RML is highly
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Fig. 1 (a) Fluorescence spectra changes of RML (10 μM) with the pH value
reducing from 7.40 to 4.40 (λex = 560 nm). Inset: the fluorescent color of
RML changes from colorless to red with the pH decreasing. (b) Sigmoidal
fitting of the fluorescence intensity at 583 nm vs pH values and the linearity
relationship over the pH range from 4.50 to 5.70.
Fig. 3 Confocal microscopy fluorescence images of 10 μM RML (a, e) and
LysoTracker Green DND-26 (15 μM) (b) and MitoTracker Green (1 μM) (f)
co-stained in HeLa cells, respectively. Merged image (c, g). The correlation
of RML with LysoTracker Green DND-26 intensities (A = 0.89) (d) and the
correlation of RML and Mitotracker green intensities (A = -0.14) (h). The
red channel image was collected at 568 - 650 nm (λex = 561 nm) for RML.
The green channel image was collected at 490 - 530 nm (λex = 488 nm) for
LysoTracker Green DND-26 and MitoTracker Green. Scale bar: 10 μm.
selective to pH changes and workable in biological matrixes
(Fig. 2a). In addition to selectivity, we also confirmed that
RML was high photostability (Fig. S3, ESI†), excellent
reversibility against pH changes (Fig. 2b) and low cytotoxicity
(Fig. S4, ESI†), respectively.
punctuated fluorescence were cell membrane permeable (Fig.
3a, e). We also calculated the logP value (n-octanol/water
partition coefficient) of RML as 5.17 using ChemBioDraw
14.0, 24,35,36 further indicating its easy membrane permeability.
More importantly, the red fluorescence image produced by
RML (Fig. 3a, e) overlapped well with that from LysoTracker
Green (Fig. 3b, c), exhibiting a Pearson's co-localization
coefficient of 0.89. Meanwhile, the poor co-stained effect of
probe with Mitotracker green (Fig. 3e-h) (A = -0.14) was
observed. These results implied that probe can selectively
accumulated in lysosomes.
To confirm the structure-property relationship of RML, 1H
NMR-spectra at various pH values were investigated. As shown
in Fig. S5 (ESI†), upon decreasing pH from 7.40 to 4.50, a
distinct down-field shift is observed for the chemical shift
values of the benzene ring protons (H3, H4 and H5) around
diethylamino and enthyl protons (H1 and H2) near spirocyclic
ring, whereas the chemical shift values of the other protons are
almost constant. These results suggest that the protonation
occurs at diethylamino N atom, and further inducing spirocyclic
ring opening, which results in the decrease of electron density
around these protons. Thus, it is clear that the protonation of
diethylamino N induces spirocyclic ring opening, leading to
remarkable fluorescence enhancement.
Lysosome-specific intracellular pH sensing using RML
RML was further investigated for its pH-dependent
fluorescence behaviour in a set of various pH high K+ buffer
with nigericin as the H+/K+ ionophore.34 Almost no
fluorescence could be observed at the neural condition of
pH7.40 (Fig. 4a). Whereas reducing the pH to pH 4.50, the red
fluorescence brightness enhanced gradually (Fig. 4b-g). In
addition, RML also exhibited the similar pH-dependent
fluorescence images in other cells such as T98G (Fig. S6, ESI†)
and SMMC-7721 cells (Fig. S7, ESI†), further confirming that
Co-localization experiment
As reported that the methylcarbitol group has the lysosome-
locating capability.24-28 We next investigated the subcellular
localization of RML by spatially matching the fluorescence of
RML (10 μM) with a commercial green-emissive tracker for
lysosomes, LysoTracker Green DND-26 (15 μM) in a co-
localization imaging experiments. As predicted, confocal
fluorescent microscopic images showed that RML with red
Fig. 2 (a) Selectivity of 10 μM RML to different potential interfering
substances in 40 mM B-R buffer solution at pH 7.40 and 4.40, respectively:
1, blank; 2, K+ (150 mM), 3, Na+ (150 mM), 4, Mg2+ (2 mM), 5, Ca2+ (2 mM),
6, Ba2+ (0.2 mM), 7, Cu2+ (0.2 mM), 8, Fe2+ (0.2 mM), 9, Fe3+ (0.2 mM), 10,
2-
Ni2+ (0.2 mM), 11, Zn2+ (0.2 mM), 12, Cl- (10 mM), 13, SO4 (0.2 mM), 14,
the probe could monitor the lysosomal pH
SO32- (0.2 mM), 15, NO- (0.2 mM), 16, Ac- (0.2 mM), 17, H2O2 (0.1 mM), 18,
ClO- (0.1 mM), 19, 1O2 (0.1 mM), 20, Cys (0.1 mM), 21, GSH (0.1 mM), 22,
Hcy (0.1 mM), 23, Ala (0.1 mM), 24, His (0.1 mM), 25, Arg (0.1 mM), 26, Lys
(0.1 mM), 27, Phe (0.1 mM), 28, Met (0.1 mM), 29, Leu (0.1 mM). (b)
Fluorescence intensity changes of RML between pH 4.40 and 7.40.
Conditions: λex = 560 nm; λem = 583 nm.
Fig. 4 Fluorescence images of HeLa cells incubated with RML (10 μM) at pH
7.40 (a), 6.00 (b), 5.65 (c), 5.35 (d), 5.00 (e), 4.75 (f) and 4.50 (g),
respectively. (h-n) Bright-field cells images of a-g. (o-u) The corresponding
merged cells images. The red emission was collected from 568 to 650 nm
(λex = 561 nm). Scale bar: 20 μm.
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a pKa value of 4.96. The linear response with pH changed from
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DOI: 10.1039/C9TB02551K
4.50-5.70, which is favorable for lysosomal pH imaging. We
further revealed that RML diffuses selectively into lysosomes
and permits the visual monitoring of autophagy by detecting the
lysosomal pH changes. Therefore, RML will be a potential
imaging tool for investigating lysosome-associated
physiological and pathological processes.
Experimental section
Materials and apparatus
Fig. 5 Visualization of autophagy using RML in HeLa cells. (a) Fluorescence
imaging of RML in HeLa cells were cultured under starvation conditions
(medium of HBSS without bovine serum for inducing cell autophagy), rich-
nutrient conditions (normal medium) and autophagy inhibited conditions
(HBSS with 3-methyladenine (3-MA, 100 μM) for inhibiting autophagy) for
a certain time (0-4 h), respectively. Cells were incubated with RML (10 μM)
for 10 min before imaging. The red emission was collected from 568 to 650
nm (λex = 561 nm). (b) Mean fluorescence intensity changes of a certain
time (0-4 h) under different conditions (Starvation, Rich-nutrient and
Starvation + 3-MA). Scale bar: 10 μm.
Rhodamine B, ethylenediamine, trimethylamine, TsCl, 2-(2-
methoxyethoxy)ethanol, MTT were obtained from Sigma-
Aldrich. Mito Tracker Green and LysoTracker Green DND-26
were commercially available from Invitrogen. Hank's balanced
salt solution (without bovine serum) was purchased from
Beijing Solarbio Science
Methyladenine were purchased from Shanghai Macklin
Biochemical Company.
& Technology Company. 3-
changes in living cells. It should be noted that the fluorescence
of probe seemed to distribute throughout the whole cytoplasmic
matrix, which has been observed in many previous
reports.32,34,37-39
NMR spectra were measured on a Bruker instrument.
High-resolution mass spectra was onbtaibed with a Thermo
Scientific Q Exactive. pH values were acquired with a Beckman
Φ50 pH meter. The UV-visible spectra measurements were
Visual monitoring the lysosomal pH changes during autophagy by
RML
Considering that during the membrane fusion process of
autophagy, the lysosomal pH in lysosomes would change due
to the micro-environmental difference between the
autophagosomes and lysosomes11, we monitored the lysosomal
pH changes during autophagy under starvation conditions.
Herein RML-stained HeLa cells were cultured in Hank’s
Balanced Salt Solution (HBSS), a medium without nutrient to
induce autophagy by starvation11, for 0 h, 1 h, 2 h, 3 h or 4 h,
respectively. As demonstrated in Fig. 5, the red fluorescence
intensities enhanced gradually along with the starvation time,
implying that the lysosomal pH has been decreased during the
autophagy process. This might due to that the pH in
autophagosomes are lower than that of lysosomes, inducing in
a slight decrease of pH in the autolysosomes during the
membrane fusion process of autophagy.40-42 For comparison,
the fluorescence intensities remained almost constant both
under under rich-nutrient conditions (normal medium) (Fig. 5a,
black line), and autophagy inhibited conditions (HBSS with 3-
methyladenine (3-MA) for inhibiting autophagy) (Fig. 5b, blue
line). Moreover, the red fluorescence emissions within the same
field of vision in HeLa cells also enhanced along with the
starvation time (Fig. S8, ESI†), further confirming that the
fluorescence change of RML could be used as an efficient
detection signal for visualization the autophagy in living cells.
obtained from
Fluorescence spectra measurements were recorded with a FLS-
920 Fluorescence Spectrometer.
a
TU-1901 UV-visible spectrometer.
Synthesis and characterization
Synthesis of Compound 1
Compound 1 was synthesized according to our previous
reported.27,28
Synthesis of Compound 2
1.00 g (2.08 mmol) rhodamine B, 2.00 mL ethylenediamine and
1.00 mL triethylamine was mixture and refluxed in 50 mL
anhydrous methanol for 24 h until the deep-red color of
rhodamine B changed to deep green. The mixture was dried
over anhydrous MgSO4, following purified using
chromatography with silica gel (CH2Cl2/CH3OH, 20:1, v/v),
affording a pink powder (0.94 g, 86 % yield). 1H NMR (CDCl3,
600 MHz) δ (ppm): 1.16-1.18 (t, 12H, -CH3-), 2.41-2.43 (t, 2H,
-CH2-), 3.19-3.21 (t, 2H, -CH2-), 3.32-3.36 (m, 8H, -CH2-),
6.27-6.29 (d, 2H, Ar-H), 6.38 (s, 2H, Ar-H), 6.43-6.45 (d, 2H,
Ar-H), 7.10 (m, 1H, Ar-H), 7.45 (m, 2H, Ar-H), 7.91 (m, 1H,
Ar-H).
Synthesis of RML
A mixture of 0.30 g (0.62 mmol) compound 1 and 0.17 g (0.62
mmol) compound 2 was refluxed in 50 mL DMF overnight.
Then mixture was dried over anhydrous MgSO4, following
purified using chromatography with silica gel (CH2Cl2),
affording a light yellow powder (0.25 g, 68 % yield). 1H NMR
(DMSO-d6, 600 MHz) δ (ppm): 1.11-1.13 (t, 12H, -CH3-), 2.44
(s, 2H, -CH2-), 2.64 (s, 2H, -CH2-), 3.32-3.30 (m, 13H, -CH2-),
3.47 (m, 4H, -CH2-), 3.52 (m, 2H, -CH2-), 6.23-6.24 (d, 2H, Ar-
H), 6.34 (s, 2H, Ar-H), 6.39-6.40 (d, 2H, Ar-H), 7.03 (m, 1H,
Ar-H), 7.39 (m, 2H, Ar-H), 7.85 (m, 1H, Ar-H). 13C NMR
Conclusion
In summary, a novel pH fluorescent probe (RML) was
presented for visual monitoring of lysosomal pH fluctuate, by
introducing methylcarbitol unit as the lysosome-targeting
group to rhodamine B, which is highly sensitive to pH changes.
Owing to the H+-induced spirocyclic ring opening, RML
exhibited a remarkable pH-dependent behavior at 583 nm with
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4
5
6
7
8
J.M. Swanlund, K.C. Kregel and T.D. Oberley, Autophagy.,
(DMSO-d6, 150 MHz) δ (ppm): 12.56, 39.81, 44.31, 48.28,
53.51, 58.91, 65.08, 69.82, 77.13, 77.35, 77.56, 97.69, 105.16,
108.06, 122.66, 123.75, 127.98, 128.62, 130.91, 132.44, 148.70,
153.21, 153.61, 168.51. HR-MS m/z: [M+H]+ calculated for
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HeLa cells were treated with RML (10 μM) for 10 min, then
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Confocal Microscope (Zeiss, LSM880+Airyscan), with red
channel (Ex = 561 nm, Em = 568 - 650 nm) for RML, and
Green channel (Ex = 488 nm, 490 - 530 nm) for Mito Tracker
Green or Lyso Tracker DND-26, respectively.
Lyso-specific intracellular pH sensing using RML
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HeLa cells were first treated with RML (10 μM) for 10 min,
then cultured in high K+ buffer (0.5 mM MgSO4, 30 mM NaCl,
120 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM NaH2PO4,
20 mM NaOAc and 20 mM HEPES) containing H+/K+
ionophore nigericin (10 μg/mL), at various pH (4.50, 4.75, 5.00,
5.35, 5.65, 6.00 and 7.40) for another 10 min, respectively.
Visual monitoring the lysosomal pH changes during autophagy
using RML
HeLa cells were first treated with RML (10 μM) for 10 min,
and then cultured under starvation conditions (HBSS without
bovine serum to induce autophagy), rich-nutrient, or autophagy
inhibited conditions (HBSS with 3-methyladenine (3-MA) (100
μM) to inhibit autophagy) for 0 - 4 h, respectively.
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Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
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Financial supports from the National Natural Science
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This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 7
Journal of Materials Chemistry B
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DOI: 10.1039/C9TB02551K
Graphical Abstract
Title: “Visual monitoring of the lysosomal pH changes during
autophagy with a red-emission pH fluorescent probe”
Authors: Xiaodong Wang†, Li Fan*†, Yubin Wang, Caihong Zhang, Wenting Liang, Shaomin
Shuang and Chuan Dong*
We report a red-emission pH fluorescent probe (RML) for visual monitoring of
the lysosomal pH changes during autophagy in living cells.