A two-photon fluorescent probe for real-time monitoring of autophagy by ultrasensitive detection of the change in lysosomal polarity

Article abstract of DOI:10.1039/c7cc00752c

The first two-photon probe, Lyso-OC, was proposed for use in monitoring cell autophagy by detection of the change in the lysosomal polarity during the membrane fusion process of autophagy. The Lyso-OC probe exhibited desirable optical properties and a det

Full text of DOI:10.1039/c7cc00752c

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DOI: 10.1039/C7CC00752C
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A two-photon fluorescent probe for real-time monitoring
autophagy by ultrasensitive detection of the change in lysosomal
polarity
Received 00th January 20xx,
Accepted 00th January 20xx
Jiacheng Jiang,a‡ Xiaohe Tian,b‡ Changzhi Xu,c Shuxin Wang,a Yan Feng,a Man Chen,a Haizhu Yu,a
DOI: 10.1039/x0xx00000x
Manzhou Zhua and Xiangming Menga*
www.rsc.org/
The first two-photon probe Lyso-OC for monitoring cell autophagy In the meantime, two-photon probes have become the leading
by detecting the change of lysosomal polarity during the bio-imaging tools thanks to their vast merits such as localized
membrane fusion process of autophagy was proposed. Lyso-OC excitation, increased penetration depth, reduced tissue
exhibited desired optical properties and selective detection signal autofluorescence and photobleaching over the one-photon
11
to the polarity change. More importantly, Lyso-OC displayed a ones.10,
Thus, we believe that detecting the change of
real-time monitoring of autophagy in living cells.
lysosomal polarity during the member fusion in autophagy
with novel two-photon fluorescent probes may be an efficient
method for monitoring autophagy (Scheme 1).
Autophagy is a lysosomal degradation pathway that primarily
serves as an adaptive agent for the protection of organisms
against diverse pathologies, including infections, cancer,
neurodegeneration, aging, and heart disease.1-3 This special
function makes the accurately monitoring technology of
autophagy in highly demand. methods have been developed
for monitoring autophagy including Transmission electron
microscopy (TEM), Western Blotting (Atg8 / LC3) and GFP-Atg8
/ LC3 fluorescence microscopy.4, 5 However, these classical
approaches are time consuming and financial costly, as well as
lack of the autophagy information in a single living cell.
Therefore, how to precisely and vividly monitoring autophagy
in a simple and cost-effective manner is still under challenge.
Autophagy is initiated by the formation of autophagosomes,
which are the organic complex that capable of engulfing
cytosolic components both selectively and non-selectively.
After the formation, autophagosomes grow and deliver
sequestered components to lysosomes.6 Lysosomes are
membraneous acidic vesicles, which contain approximately 50
different degradative enzymes and proteins that are active at
acidic pH.7 The polarity of lysosome changed greatly during the
member fusion process in autophagy due to the inner micro-
Herein, we reported the first two-photon lysosome-targeting
probe
Lyso-OC
(7-((4-methoxyphenyl)
ethynyl)-N-(2-
for
morpholinoethyl)-2-oxo-2H-chromene-3-carboxamide)
monitoring autophagy by detecting the change of lysosomal
polarity during autophagy process. Coumarin was used as the
fluorescent group for its solvatochromic property, i.e. it can
responds fluorescently to the change of micro-environmental
polarity for the variations in the molecular dipole upon
excitation.12-15 Morpholine was used as the lysosomal targeting
group for its excellent lysosome targeting property.16 To
achieve the large two-photon absorption cross-section and
Stokes shift, 1-ethynyl-4-methoxybenzene was grafted at the
7-position of the coumarin group (Scheme 1).17, 18 We hope
Lyso-OC will give
environmental
difference
between
lysosome
and
autophagosome.8-10
a.Department of Chemistry, Anhui University, Hefei, Anhui, 230601 (China)
b.School of Life Sciences, Anhui University, Hefei, Anhui, 230601 (China)
c. Institute of Health Sciences, Anhui University, Hefei, Anhui, 230601 (China)
* Correspondence: E-mail: mengxm@ahu.edu.cn
‡
These authors contributed equally to this work.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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fluorescence intensity of Lyso-OC was depended on solvent
polarity (Figure S1, S2 and Table S1). However, the change of
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DOI: 10.1039/C7CC00752C
Scheme 1. The structure of Lyso-OC and the mechanism of
monitoring autophagy by Lyso-OC in living cells.
Figure 2. a) Fluorescence imaging of MCF-7 cells co-stained
with Lyso-OC (10.0 μM) and Lyso Tracker Red (0.5 μM) for 45
min. b) Fuorescence imaging of MCF-7 cells stained with Lyso-
OC (10 μM) for 45 min. c) Fluorescence imaging of MCF-7 cells
in b) further treated with DMSO (10.0 μL) for 60 min, λex = 760
nm, λem = 490-550 nm.
the solvent polarity caused negligible effect on the absorption
of the probe (Figure S3). The calculation results also confirmed
the observation data (Figure S4 and S5). These results suggest
that the fluorescence of Lyso-OC is highly sensitive to the
change of micro-environmental polarity. We further examined
the effect of solvent polarity on the quantum yield and lifetime
of Lyso-OC in H2O/THF mixtures. The quantum yield and
lifetime values showed remarkable linearity with Δƒ from 0.256
to 0.313 (Figure S6 and S7), in good accordance with the
emission behavior. Similar behavior of Lyso-OC was observed
in THF/MeOH mixtures (Figure S8). These data confirmed that
the fluorescence of Lyso-OC was regulated by polarity of the
solvent.21-23
Figure 2. a) Fluorescence emission spectra of Lyso-OC (10 μM) in
different H2O/THF mixtures (water from 10 % to 80 %). Insert:
Linearity of Imax versus the solvent parameter Δƒ. b) Two photon
action cross sections of Lyso-OC in different H2O/THF mixtures
(water from 10 % to 80 %). Insert: The square relationship of two-
photon excited fluorescence intensity (Iout) of Lyso-OC with the
addition of input power (Iin = 300-800 mW). λex = 375 nm.
The TP cross sections of Lyso-OC were determined by the two-
25
photon induced fluorescence technique.24,
As shown in
Figure 1b, the two-photon action cross-section (Φδ) of Lyso-
OC at 760 nm decreased gradually from 102 GM (10% water)
to 4.4 GM (80% water) in H2O/THF mixtures. Furthermore,
with the increase of laser power from 0.2 W to 0.8 W at 760
nm, a square relationship of two-photon excited fluorescence
intensity is observed with a reasonable linearity, indicating the
two-photon excitation nature of the probe.
In the pH titration and viscosity titration experiments, the
fluorescence intensity of Lyso-OC is found to be pH-insensitive
in the lysosomal pH range (Figure S9) and viscosity-insensitive
(Figure S10). MTT assay results indicated that the new probe is
low cytotoxicity and suitable for detection polarity change in
living cells (Figure S11).26 To further investigate the subcellular
good two photon fluorescent detection signal to the change of
lysosomal polarity during the autophagy process.
The emission of Lyso-OC under different solvent polarity was
tested in H2O/THF system. Lippert Mataga polarity parameter
Δƒ was used to indicate the polarity of the solution.19, 20 As
shown in Figure 1a, when the solvent changed from 10% water
(Δƒ
≈ 0.256) to 80% water (Δƒ ≈ 0.313) in H2O/THF mixtures,
the fluorescence intensity of Lyso-OC decreased about 40-fold
along with a red shift (~ 30 nm) of the emission spectra. A
good linear correlation between the fluorescence intensity
maximum and solvent polarity (Δƒ) is obtained. Optical
properties of Lyso-OC in different solvents also confirmed that
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localization of Lyso-OC, co-localization experiment with Lyso in the autophagy process. The rate of GAPDH and LC3-II could
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DOI: 10.1039/C7CC00752C
Tracker Red was carried out in MCF-7 and HeLa cells. As shown be used to reflect the level of autophagy. As shown in Figure
Figure 4. a) Fluorescence confocal imaging of MCF-7 cells
stained with Lyso-OC before be induced. b) Fluorescence
Figure 3. Representive electron microscopic images of the
cytoplasmic regions of MCF-7 cells. Cells were cultured in
starvation for different times and stained with osmium
tetroxide. n = nucleus, lyso = lysosome, AVi = autophagosome,
AVd = degradative autophagic vacuole, IV = internal vacuoles.
confocal imaging of MCF-7 cells stained with Lyso-OC in
autophagy. λex = 760 nm, λem = 490-550 nm. The scale bar
represents 20 μm.
S13, the level of LC3-II has increased obviously during the early
stages of autophagy in MCF-7 cells and reached the maximum
after incubating for 1 h. The results demonstrate that the
autophagy of MCF-7 cells could be induced by starvation and
the membrane fusion process began within one hour.
in Figure 2 and Figure S12, the images of Lyso-OC and Lyso
Tracker Red overlapped very well with each other. co-
localization coefficient was calculated to be 0.91 and 0.89 in
MCF-7 and HeLa cells respectively which suggested that Lyso-
OC localized in lysosomes in living cells.27 The three-
dimensional fluorescence imaging of rat liver tissue slices
incubated with Lyso-OC indicated that the penetrative depth
of the probe was 120 μm under the two-photon fluorescence
excitation (Figure S13).28
The change of lysosomal polarity in autophagy was further
investigated. As shown in Figure 4a, Lyso-OC distributes in the
The ability of Lyso-OC to detect the change of lysosomal
polarity was further tested. We examined Lyso-OC staining in
multiple cell lines including MCF-7 cells, HeLa cells, HELF cells
and CHO cells treated with dimethyl sulfoxide (DMSO), which
can be absorbed by cells in a short time.17 As shown in Figure 2
and Figure S14, it was found that the fluorescence of cells has
increased obviously after adding 10 μL DMSO to the cell
culture medium within 60 minutes. The sensitivity to polarity
of Lyso-OC in lysosome encourages us to monitor cell
autophagy by detection of the change of lysosomal polarity
during the autophagy process with Lyso-OC
.
Hank's Balanced Salt Solution (HBSS), a medium without
nutrient, was used to induce autophagy of MCF-7 cells by
starvation. Transmission electron microscopy (TEM) was
eventually adopted to monitor the autophagy induced by
starvation at subcellular level.29, 30 As shown in Figure 3, in cells
with rich nutrient, the normal cytoplasm and lysosomes can be
observed vividly. However, after incubated with HBSS for 1
hour, the formation of autophagosomes (AVi) was clearly
observed and the membrane fusion between lysosomes and
autophagosomes can already been observed. Then,
autolysosomes (AVd) and the region filled by small internal
Figure 5. a) Real-time confocal imaging of MCF-7 cells in rich-
vesicles could be observed at 4 hours. Western blotting, nutrient (control), starvation (autophagy) and starvation + 3-
MA (autophagy inhibited), respectively. Cells were stained
with Lyso-OC (10.0 μM) for 45 min before imaging. b)
Fluorescence intensity ratio (I / I0) of Lyso-OC in rich-nutrient
(black line), starvation (red line) and starvation + 3-MA (blue
line) at each time nodes. I represents the intensity at each
another classical way for detecting autophagy, was carried out
simultaneously. The expression level of LC3-II protein in cells is
regarded as marker of autophagy.4, 5 GAPDH, one of the key
enzymes involved in glycolysis, is kept in the original quantity
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With the above data in hand, we tried to use Lyso-OC to
monitor autophagy with fluorescent signal. The con-focal
imaging of the MCF-7 cells stained with Lyso-OC was recorded
at different time (0-4 hours) under different cell culture
conditions (normal, autophagy and autophagy inhibited). As
shown in figure 5, the fluorescence of cells in rich nutrient
(normal) has kept constant at different time dues (Video S1)
suggesting probe Lyso-OC is photo resistance at this
experimental circumstance. However, the fluorescence of cells
in starvation decreased to 40% gradually with time increase
(Video S2). Furthermore, cells were incubated in HBSS in the
presence of 3-Methyladenine (3-MA, autophagy inhibitor), the
fluorescence in these cells was found to be remained
unchanged with the incubation time increasing (Video S3).
From Figure 5 and Video S2, we can also see that the
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could be recorded vividly. Similar results were found in HeLa
cells (figure S16).Thus, the fluorescent change of Lyso-OC
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cells, HELF cells and CHO cells demonstrates the detecting
ability and wide applicability of Lyso-OC to lysosomal polarity.
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cell and multiple cells by detecting the change of lysosomal
polarity efficiently and economically. These results provide
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