A dual-site controlled ratiometric probe revealing the simultaneous down-regulation of pH in lysosomes and cytoplasm during autophagy

Article abstract of DOI:10.1039/c9cc03679b

In this work, a unique dual-site controlled fluorescent probe was presented for the sensitive and concurrent detection of pH in the cytoplasm and lysosomes. With the probe, the simultaneous down-regulation of pH in the lysosomes and cytoplasm during autophagy has been successfully revealed for the first time.

Full text of DOI:10.1039/c9cc03679b

View Article Online
View Journal
ChemComm
Chemical Communications
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Tian, C. Liu, B.
Dong, Y. Zuo and W. Lin, Chem. Commun., 2019, DOI: 10.1039/C9CC03679B.
Volume 54
Number
January 2018
Pages 1-112
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
1
4
ChemComm
Chemical Communications
rsc.li/chemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
towards bifunctional catalysts of tunable basicity
rsc.li/chemcomm

Page 1 of 4
Please d oC hn eo mt Ca do mj u ms t margins
View Article Online
DOI: 10.1039/C9CC03679B
COMMUNICATION
A dual-site controlled ratiometric probe revealing the
simultaneous down-regulation of pH in lysosomes and cytoplasm
during autophagy
Received 00th January 20xx,
Accepted 00th January 20xx
Minggang Tian, Chuang Liu, Baoli Dong, Yujing Zuo, and Weiying Lin*
DOI: 10.1039/x0xx00000x
In this work, a unique dual-site controlled fluorescent probe was the fluorescence imaging of intracellular pH, many kinds of
presented for the sensitive and concurrent detection of pH in fluorescent probes have been constructed with various
1
2-13
cytoplasm and lysosomes. With the probe, the simultaneous down- response sites and fluorescence mechanisms.
For example,
regulation of pH in lysosomes and cytoplasm during autophagy has Urano et al. have presented a near-infrared fluorescent probe
been successfully revealed for the first time.
for pH in acid compartments using piperazine as the response
group, working on photo-induced electron transfer (PET)
mechanism. Kim and co-workers have developed fluorescent
Autophagy is a significant pathway for live cells to degrade
unwanted components including proteins and organelles.
14
1
probes for ratiometric detection of lysosomal pH based on
Autophagy has attracted intense focus in recent years, owning
to its indispensable roles in infection, self-repairing,
programmed cell death, and some other biological and
15
imidazole group. Yu et al. have reported a fluorescent probe
for mitochondrial pH with a hydroxyl group as the recognition
16
site.
2
-3
pathological processes. Abnormal regulation of autophagy is
implicated in many severe diseases such as Pakinson disease
and cancer.4 During the autophagy process, the substrates in
-5
6
cytoplasm are transported into lysosomes and digested.
Lysosomal and cytoplasm pH plays complex roles in autophagy
process, and for example, the raise of lysosomal pH could
efficiently inhibit autophagy by decreasing the activity of
hydrolase.7 Researches also prove that the acidic pH
8
environments can induce and promote the autophagy levels.
Therefore, monitoring pH changes in cytoplasm and lysosomes
during autophagy is of fundamental significance for the
investigation on the internal relationship between pH and
autophagy, and promoting the in-depth understanding of
autophagy.
Nowadays, colormetric analysis, electrochemical, and other
methods have been applied in the detection of pH values. In synthetic routine of Cyto-Lyso (b).
Scheme 1. The sensing mechanism of Cyto-Lyso to pH in neutral and acid ranges (a); the
9
-10
comparison with these techniques, fluorescence microscopy
1
1
displays superior advantages. With suitable fluorescent
probes, the intracellular pH could be mapped at micrometer
scale. The dynamic pH changes in live cells can be in-situ and
real-time visualized under fluorescence microscope. To realize
To investigate the pH change in cytoplasm and lysosomes
during autophagy, fluorescent probes targeting both lysosomes
and cytoplasm and sensitive to pH in neutral and acid range are
in urgent need. A possible strategy is to develop dual-site
controlled pH probes. Tang et al. have reported a dual-site
fluorescent probe for intracellular pH values, but the probe was
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of insensitive to pH in the acid range (4.5-5.5), which limited its
Jinan, Jinan, Shandong 250022, People’s Republic of China. E-mail:
weiyinglin2013@163.com.
Electronic Supplementary Information (ESI) available: [Synthesis of the probe,
absorption and fluorescence spectra, characterization data]. See
DOI: 10.1039/x0xx00000x
17
application in detecting lysosomal pH. Our group has also
designed a dual-site fluorescent probe for ratiometric detection
of lysosomal pH. The probe exclusively target lysosomes due
18
Please do not adjust margins

Please d oC hn eo mt Ca do mj u ms t margins
Page 2 of 4
COMMUNICATION
Journal Name
to the basic properties of morpholine and piperazine, and thus ratiometrically image the pH change in acid range oVifewlyAsroticsloe mOnelinse.
cannot be used to image cytoplasm pH. Up to now, fluorescent Moreover, the intensity ratio decreasedDOfrIo: 1m0.1003.091/C29CtoC003.60709B4
probes enabling the simultaneous visualization of pH in when the pH changed from 6.0 to 8.0, indicating that Cyto-Lyso
cytoplasm and lysosomes have not been reported yet.
can detect pH change in neutral range of cytoplasm.
Consequently, the probe should exhibit two sites to sense the
The interferences from various bioreagents including heavy
pH in neutral and acid range for cytoplasm and lysosomes, metals, biological ions, phosphates, etc. have been testified as
respectively, and distributed in lysosomes and cytoplasm. In this shown in Figure S1. Obviously, these reagents would not induce
work, as presented in Scheme 1a, 7-hydroxyl-coumarin moiety evident change to the fluorescence of Cyto-Lyso, confirming the
was selected for the neutral pH range, and amino-rhodamine high selectivity of the probe to pH values.
part was used for the acid pH. Meanwhile, amide group with
The cytotoxicity of Cyto-Lyso was consequently testified
weak basic properties was decorated onto the probe, to ensure before the cell imaging experiments. As shown in Figure S2a, the
that the probe can target both lysosomes and cytoplasm. As a survival rates of HepG2 cells incubated with 5 μM Cyto-Lyso for
result, the probe Cyto-Lyso targets both cytoplasm and 2 h, 12 h, and 24 h are 99.5%, 96.0%, and 94.3%, respectively.
lysosomes, and enables the ratiometric detection of pH in the Moreover, the viability of live cells incubated with 5-30 μM
two locations with high sensitivity. The down-regulation of pH Cyto-Lyso for 12 h is above 85 % (Figure S2b), indicating the low
values in cytoplasm and lysosomes have been successfully cytotoxicity of the probe. In cell imaging experiments, live
revealed, and the inhibition of NH Cl and chloroquine (CQ) to HepG2 cells were incubated with 5 μM Cyto-Lyso for 30 min,
4
autophagy has been evidently observed by means of the probe. and the cytotoxicity could be ignored under the experimental
conditions.
The photostability of Cyto-Lyso in cells was also investigated
in-situ with the ceaseless irradiation of 405 nm and 561 nm
lasers. In Figure S3, after the irradiation for 16 min, intense
intracellular emission was still detected and the fluorescence
bleaching could be ignored. These results demonstrated the
high photostability of Cyto-Lyso, which was potential for the
long-term imaging of live cells.
Figure 1. The emission spectra of 10 μM Cyto-Lyso under different pH values with the
excitation of 405 nm (a) and 500 nm (b); the pH-dependent fluorescence intensity at 455
nm (excited by 405 nm) and 588 nm (excited by 500 nm) (c); the pH-dependent intensity
ratio of 588 nm to 455 nm, insert: amplification at pH values of 6.0 ~ 8.0.
Figure 2. The fluorescence images of live HepG2 cells incubated with 5 μM Cyto-
The optical properties of Cyto-Lyso were acquired initially to Lyso for 30 min (a-c) or co-stained with 5 μM Cyto-Lyso and 200 nM LTDR for 30
min (d-f); the co-localization scatter plot (g); the intensity distribution of Cyto-Lyso
and LTDR along the arrow in Figure 2f. Bar = 20 μm.
confirm its response to different pH values. As depicted in
Figure 1a, Cyto-Lyso displayed very weak blue emission at the
pH of 3, and the fluorescence intensity sharply increased by ~
Live HepG2 cells were then incubated and imaged with the
2
30-fold when the pH value changed from 3.0 to 8.0.
probe Cyto-Lyso, as shown in Figure 2a-2c. Evidently, Cyto-Lyso
displayed intense blue and weak red fluorescence in live HepG2
cells. Observed from the merged images, the red and blue
fluorescent signals localized in different compartments in cells.
The red signals presented in small dots which is in accordance
with the lysosomal morphology. Considering that strong red
emission only exists in acid conditions, the red signals may
localized in lysosomes. To confirm this speculation, the red
Meanwhile, Cyto-Lyso showed strong red fluorescence at the
pH of 3.0, which was nearly disappeared when the pH was
changed to 8.0. The pH-dependent fluorescence intensity at 455
nm and 588 nm was plotted in Figure 1c. From the results, the
pK
a
values of the two response parts were calculated as 3.5 and
6
.2, respectively. Consequently, the two sensing sites could be
used to assess the pH values at acid and neutral range,
respectively. To testify the ability of Cyto-Lyso in ratiometric
detection of pH values, the red-to-blue intensity ratio was also
calculated, as plotted in Figure 1d. Since the physiological range
generally distributed in the range of 4.5~8.0, the pH-dependent
ratio was plotted in the range. As displayed in Figure 1d and the
insert, the intensity ratio decreased from 1.76 to 0.04 when the
pH changed from 4.5 to 5.5, implying the ability of Cyto-Lyso to
signals of Cyto-Lyso were co-localized with LTDR,
a
commercialized fluorescent probe for lysosomes. As presented
in Figure 2d-2f, the red signals of Cyto-Lyso overlapped well
with the LTDR shown in green pseudo color, and the co-
localization coefficient was up to 0.89. Moreover, according to
the scattering plot and spatial distribution of Cyto-Lyso and
LTDR, the two probes co-localized well in live HepG2 cells. Thus,
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
Please d oC hn eo mt Ca do mj u ms t margins
Journal Name
COMMUNICATION
red signals from Cyto-Lyso actually localized in lysosomes. presented in Figure S6, the Atg-7 protein overexpVrieewssAertdicleuOnndliener
Moreover, in Figure 2a, the blue signals from Cyto-Lyso the starvation for 2 h, indicating the activDaOtiIo: 1n0.o10f3a9u/Cto9pChCa0g36y7.9InB
distributed in the cytoplasm. Co-localization experiments of Figure 3a, HepG2 cells in the culture medium showed intense
Cyto-Lyso with ERTR and MTDR, commercial probes for fluorescence in blue channel, and weak emission in red channel.
endoplasmic reticulum (ER) and mitochondria, were performed. By contrast, the cells after the starvation for 60 min showed
In Figure S4, the blue signals of Cyto-Lyso well overlapped with slightly decreased blue emission, and largely enhanced red
ERTR and partially overlapped with MTDR, and the Pearson’s emission, indicating the slightly decreased pH in cytoplasm and
coefficients were 0.85 and 0.65, respectively. These results largely decreased pH in lysosomes, respectively. The pH
indicate that Cyto-Lyso mainly localized in ER. The ER changes could be clearly observed from the ratiometric images.
1
9
membrane is highly permeable to protons, and ER pH is the According to the results in Figure S5, the cytoplasm pH
same as the cytoplasm. Consequently, Cyto-Lyso could image decreased to ~7.0, and the lysosomal pH decreased to ~5.0. The
cytoplasm pH.
cells after the starvation for 120 min were also imaged with
The response of Cyto-Lyso to intracellular pH was then Cyto-Lyso. In Figure 3a, compared with the cells starved for 60
confirmed. Live HepG2 cells were incubated by culture medium min, these cells showed evidently decreased emission in the
with different pH values. The nutritious culture medium can blue channel, and further enhanced fluorescence in the red
block the autophagy, and the different pH of the culture channel. Ratiometric images were also acquired, and the
medium would alter the intracellular pH values. In Figure S5, cytoplasm pH was in the range of 6.0~7.0 in these cells, while
compared with the cells cultured in normal culture medium (pH the lysosomal pH was in 4.0~5.0. These results demonstrated
7
.2-7.4), cells treated at the pH of 8.0 showed evidently that both the cytoplasm pH and lysosomal pH actually
enhanced emission in blue channel and decreased fluorescence decreased during the autophagy process.To clearly assess the
in red channel, indicating that Cyto-Lyso could image the arised pH changes in cytoplasm and lysosomes during autophagy, the
pH in cells. In comparison, the cells treated in the pH of 7.0, 6.0, mean intensity in blue and red channels and their ratio have
5
.0, and 4.0 displayed gradually decreased blue fluorescence been calculated in Figure 3b. The decreased blue emission
and enhanced red emission, confirming that Cyto-Lyso could intensity could be evidently observed, and the red to blue ratio
image the pH changes in cells. Ratiometric images were also was also dramatically changed during the autophagy process.
obtained, and the pseudo colors were given according to the These results also demonstrated the decreased pH in neutral
color bar. In Figure S5, with the pH of the culture medium and acid range during autophagy.
changing from 8.0 to 4.0, the pseudo colors in the ratiometric
The pH changes in 4T1 cells during autophagy were also
image changed from green to red, demonstrating the increase investigated with the probe Cyto-Lyso to check the universality,
of the ratio. Therefore, Cyto-Lyso can image intracellular pH as displayed in Figure S7. Similar to the HepG2 cells, 4T1 cells
changes in a ratiometric manner.
cultured in the culture medium showed intense blue
fluorescence and weak red emission. After the starvation for 60
min, the fluorescence in the blue channel slightly decreased,
and the emission in the red channel evidently increased. In
comparison the results in Figure S5, the cytoplasm pH was
around 7.0, and the lysosomal pH was in the range of 5.0~6.0.
The starvation time was also prolonged, and obviously
decreased blue emission and further increased red fluorescence
could be observed. The cytoplasm pH was in the range of
6
.0~7.0, and the lysosomal pH changed to ~5.0. Therefore, the
cytoplasm and lysosomal pH of 4T1 cells decreased during the
starvation induced procedure.The mean intensity in two
channels and their ratio were also calculated as presented in
Figure S7b. The decreased emission in blue channel and
enhanced fluorescence in red channel could be clearly observed.
Moreover, the red to blue ratio in 4T1 cells steadily increased
during starvation. These results also demonstrated the
Figure 3. The fluorescent images of HepG2 cells pre-stained with 5 μM Cyto-Lyso for 30
min (a) then incubated with culture medium for 120 min (Control) and PBS buffer for 60
min and 120 min. The mean intensity in blue channel, red channel, and the intensity ratio decreased pH in both neutral and acid range during autophagy.
2
2
of red to blue channel of the three groups of cells. Bar = 20 μm.
CQ was reported as an inhibitor of the autophagy process,
and the effect of CQ on autophagy was investigated. As shown
The pH changes in cytoplasm and lysosomes during autophagy in Figure 4, after the starvation treatment in buffer containing
were afterwards investigated with the probe Cyto-Lyso. 25 μM CQ, the autophagy has been slightly suppressed.
2
0,21
Nutrient starvation conditions can induce the autophagy,
Meanwhile, the weak emission in cytoplasm indicating the
and therefore live HepG2 cells were stained with the probe then decreased pH in cytoplasm during autophagy. The lysosomal pH
incubated by PBS buffer for 60 min and 120 min, respectively. increased compared with the cells treated in buffer without CQ
Western Blotting (WB) experiments were performed to confirm for 120 min (Figure 3), which should be attributed to the CQ-
the activation of autophagy under starvation conditions. As caused lysosomal alkalization. With the increase of CQ
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please d oC hn eo mt Ca do mj u ms t margins
Page 4 of 4
COMMUNICATION
Journal Name
concentration, the proportion of lysosomes obviously
decreased, indicating evident inhibition of autophagy. Notably,
the cytoplasm emission obviously enhanced with the elevated
CQ concentration, indicating that the increase of cytoplasm pH
is accompanied with autophagy inhibition.
Conflicts of interest
There are no conflicts to declare.
View Article Online
DOI: 10.1039/C9CC03679B
Acknowledgement
For financial support, we thank the National Natural Science
Foundation of China (NSFC) (21472067, 21672083, 21877048,
2
1804052), the Taishan Scholar Foundation (TS201511041), the
Natural Science Foundation of Shandong Province
(
(
ZR2018BB058), and the startup funds of University of Jinan
309-10004, 160100331).
Notes and references
1
2
Mizushima, N.; Komatsu, M., Cell 2011, 147 (4), 728-741.
Thurston, T. L. M.; Wandel, M. P.; von Muhlinen, N.; Foeglein,
Á.; Randow, F., Nature 2012, 482, 414.
3
4
Cuervo, A. M.; Bergamini, E.; Brunk, U. T.; Dröge, W.; Ffrench,
M.; Terman, A., Autophagy 2005, 1 (3), 131-140.
Kitada, T.; Asakawa, S.; Hattori, N.; Matsumine, H.;
Yamamura, Y.; Minoshima, S.; Yokochi, M.; Mizuno, Y.;
Shimizu, N., Nature 1998, 392, 605.
Jin, S.; White, E., Autophagy 2007, 3 (1), 28-31.
Eskelinen, E.-L.; Saftig, P., Biochim. Biophysic. Acta 2009, 1793
Figure 4. The fluorescence images of HepG2 cells pre-stained with 5 μM Cyto-Lyso for 30
min (a) then incubated in PBS containing different amount of CQ for 2 h. Bar = 20 μm.
5
6
NH
4
3
Cl has been reported to cause the increase of intracellular
2
pH, and the effect of NH
4
Cl on autophagy was also studied in
(
4), 664-673.
Figure S8. Compared with the cells starved for 2 h, addition of
0 μM NH Cl would not bring obvious effect on autophagy.
However, the cytoplasm pH is apparently increased compared
with the groups without NH Cl. Consequently, 50 μM NH Cl can
induce pH elevation in cytoplasm, but cannot inhibit autophagy.
In comparison, 100 μM or 200 μM NH Cl could efficiently inhibit
the autophagy, and caused the increase of cytoplasm and
lysosomal pH. WB experiments were performed to 11 Chen, H.; Dong, B.; Tang, Y.; Lin, W.,. Acc. Chem. Res. 2017, 50
7
8
9
1
Filomeni, G.; De Zio, D.; Cecconi, F., Cell Death Differentiation
014, 22, 377.
5
4
2
Devin M. W.; Ju-hyun L.; Asok K.; Sooyeon L.; Samantha J. O.;
Ralph A. N.,. Eur. J. Neur. 2013, 37 (22), 1949-1961.
4
4
Wang, Y.; Tang, B.; Zhang, S., Dyes Pigments 2011, 91 (3), 294-
2
97.
4
0 Zhou, J.; Zhang, L.; Tian, Y., Anal. Chem. 2016, 88 (4), 2113-
118.
2
(6), 1410-1422.
demonstrate the inhibition of NH
the expression of Atg-7 protein of cells treated by 100 μM NH
4
Cl to autophagy. In Figure S6,
Cl
1
2 Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.;
4
Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S., J. Am. Chem. Soc.
decreased, compared with cells starved in PBS buffer, indicating
the inhibition of autophagy process. Overall, the cytoplasm and
lysosomal pH is decreased during autophagy. The cytoplasm
alkalization brings little influence on autophagy, while the
elevated lysosomal pH may inhibit autophagy.
2
014, 136 (40), 14136-14142.
1
1
3 Hou, J.-T.; Ren, W. X.; Li, K.; Seo, J.; Sharma, A.; Yu, X.-Q.; Kim,
J. S., Chem. Soc. Rev. 2017, 46 (8), 2076-2090.
4 Takahashi, S.; Kagami, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.;
Ueno, T.; Uchiyama, M.; Koyama-Honda, I.; Mizushima, N.;
Taguchi, T.; Arai, H.; Nagano, T.; Urano, Y., J. Am. Chem. Soc.
2
018, 140 (18), 5925-5933.
5 Kim, H. J.; Heo, C. H.; Kim, H. M., J. Am. Chem. Soc. 2013, 135
47), 17969-17977.
1
1
1
Conclusions
(
6 Wu, M.-Y.; Li, K.; Liu, Y.-H.; Yu, K.-K.; Xie, Y.-M.; Zhou, X.-D.; Yu,
X.-Q., Biomaterials 2015, 53, 669-678.
In summary, a dual-site fluorescent probe (Cyto-Lyso) for the
ratiometric imaging of cytoplasm and lysosomal pH values with
high sensitivity has been constructed. For molecular design, 7-
hydroxyl-coumarin moiety was used to detect cytoplasm pH,
and amino-rhodamine was utilized to sense lysosomal pH. The
probe target both lysosomes and cytoplasm, and could image
pH in the dual targets with high sensitivity. The concurrently
down-regulation of pH in cytoplasm and lysosomes was
7 Chen, S.; Hong, Y.; Liu, Y.; Liu, J.; Leung, C. W. T.; Li, M.; Kwok,
R. T. K.; Zhao, E.; Lam, J. W. Y.; Yu, Y.; Tang, B. Z., J. Am. Chem.
Soc. 2013, 135 (13), 4926-4929.
8 Dong, B.; Song, X.; Wang, C.; Kong, X.; Tang, Y.; Lin, W., Anal.
Chem. 2016, 88 (7), 4085-4091.
9 Kim, J.; Johannes, L.; Goud, B.; Antony, C.; Lingwood, C.;
Daneman, R.; Grinstein, S., Proc. Natl. Acad. Sci. 1997, 95,
2997-3002.
1
1
successfully revealed in a ratiometric manner during the 20 Mizushima, N.; Yamamoto, A.; Matsui, M.; Yoshimori, T.;
Ohsumi, Y., Mol. Biol. Cell 2004, 15 (3), 1101-1111.
1 Jiang, J.; Tian, X.; Xu, C.; Wang, S.; Feng, Y.; Chen, M.; Yu, H.;
Zhu, M.; Meng, X., Chem. Commun. 2017, 53, 3645-3648.
2 Liang, D. H.; Choi, D. S.; Ensor, J. E.; Kaipparettu, B. A.; Bass, B.
L.; Chang, J. C., Cancer Lett. 2016, 376 (2), 249-258.
4
starvation induced autophagy. The inhibition effect of NH Cl
2
2
and CQ on autophagy have been also investigated with the
probe. The probe can serve as a valid tool for the assessment of
pH changes in cytoplasm and lysosomes simultaneously, and
facilitate the fundamental researches on autophagy and 23 Hagag, N.; Lacal, J. C.; Graber, M.; Aaronson, S.; Viola, M. V.,
relative areas.
Mol. Cell. Biol. 1987, 7 (5), 1984-1988.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins