Real-time tracking of lipid droplets interactions with other organelles by a high signal/noise probe

Article abstract of DOI:10.1016/j.dyepig.2021.109366

Investigating the interactions between lipid droplets (LDs) and other organelles are greatly significant to the in-depth understand the physiology and pathology of LDs. Despite of commercial LDs probes available, the ER and other membrane structures are a

Full text of DOI:10.1016/j.dyepig.2021.109366

Dyes and Pigments 191 (2021) 109366
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Real-time tracking of lipid droplets interactions with other organelles by a
high signal/noise probe
a
,
1
a
,
1
a
,
1
a
a
a
Wei Ren , Dong Wang , Wei Huang , Jiajia Li , Xiaohe Tian , Zhengjie Liu ,
a
c
c
a,
b
a,b,*
Guangmei Han , Bianhua Liu , Ming-Yong Han , Zhongping Zhang , Ruilong Zhang
a
School of Chemistry and Chemical Engineering, And Institute of Physical Science and Information Technology, Anhui University, Hefei, 230601, China
Key Laboratory of Structure and Functional Regulation of Hybrid Materials (Anhui University), Ministry of Education, Hefei, Anhui, 230601, China
b
c
Key Lab of Photovoltaic and Energy Conservation Materials, Institute of Solid State Physics, HFIPS, Chinese Academy of Sciences, Hefei, 230031, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Investigating the interactions between lipid droplets (LDs) and other organelles are greatly significant to the in-
depth understand the physiology and pathology of LDs. Despite of commercial LDs probes available, the ER and
other membrane structures are also stained due to their amphiphilicity, which limits their further applications.
To address this issue, we herein report a highly hydrophobic fluorescent probe to specifically label intracellular
LDs with high signal/noise ratio, avoiding undesirable staining of ER and thus achieving to real-time monitor the
interactions between LDs and other organelles. The probe showed strong emission in lipid environments but no
fluorescence in water and liposomes, enabling the probe with high signal/noise ratio. Using this probe, it was
found that the contacts of LDs with ER and mitochondria clearly increased under the condition of starvation.
Hydrophobic probe
High signal/noise
Lipid droplets
Organelles interaction
1
. Introduction
Lipid droplets (LDs) are key organelles in mammalian cells, which
understand the physiology and pathology of LDs [18].
The traditional method of observing LDs is transmission electron
microscopy (TEM) [3,19,20], which has high resolution to show the
ultrafine structure but incapable of distinguishing multiple lipid envi-
ronments and tracking the dynamic of LDs in live cells. In this context,
fluorescent imaging with high sensitivity and selectivity has become a
powerful tool to real-time track LDs’ behaviors [21,22]. To date, a large
number of probes have been exploited and fabricated to label LDs
[23–25], and parts of them have already been commercialized, e. g.
BODPY 493/503 and Nile Red. However, most of the probes can also
stain other hydrophobic environments such as phospholipid layer,
making the investigation of the communication between LDs and other
organelles difficult. Therefore, improving LDs-specific fluorescent
probes with signal/noise ratio is critical to address this issue.
not only store most of lipids in living cells [1], but also exchange fat
molecules to mitochondria [2,3], endoplasmic reticulum (ER) [4–8],
and other membrane-structural organelles, especially under starvation
or drug stimulation. In LDs, there is a hydrophobic phase of neutral
lipids as the core covered by a monolayer membrane of phospholipids
[
9,10]. On the membrane of LDs, various lipoproteins are embedded to
catalyze the lipolysis [11–13] and participate in the interactions be-
tween LDs and other organelles [5,14]. For instance, upon cell starva-
tion, triacylglycerol (TG) in LDs is rapidly hydrolyzed to supply fatty
acids to mitochondria where the energy is generated for the metabolisms
of cells [15]. Under the nutrient-rich condition, TG accumulates in
phospholipid layer of ER and forms a bent ER surface, and then is
transported into LDs or forms a new LD [16]. In the case of ER stress, the
LDs can also provide the raw materials to ER for phospholipid synthesis
such as diacylglycerol (DG), to supply the consumption of the phos-
pholipid in the process of ER autophagy [17]. Accordingly, the study of
LD communication with other organelles is greatly significant to deeply
Herein, we report a highly hydrophobic probe which exhibits ag-
gregation caused quenching (ACQ) to specifically label intracellular LDs
(Scheme 1). The probe showed negligible fluorescence in water and li-
posomes, and thus avoided the staining of other membrane-structural
organelles, realizing the real-time tracking of the interactions between
LDs and other organelles. Further, we have demonstrated the contacts of
*
Corresponding author. School of Chemistry and Chemical Engineering, and Institute of Physical Science and Information Technology, Anhui University, Hefei
2
30601, China.
E-mail address: zrl@ahu.edu.cn (R. Zhang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.dyepig.2021.109366
Received 23 February 2021; Received in revised form 30 March 2021; Accepted 1 April 2021
Available online 3 April 2021
0
143-7208/© 2021 Elsevier Ltd. All rights reserved.

W. Ren et al.
Dyes and Pigments 191 (2021) 109366
water, and further purified by column chromatography eluent with
CH
2 2
Cl : MeOH (25:1, v/v). Finally, the orange solid was obtained as the
1
final product, DMQ (0.19 g, yield 65%). H NMR (400 MHz, CDCl
3
) δ
.11 (dd, J = 9.0, 14.1 Hz, 4H), 8.01 (d, J = 16.6 Hz, 2H), 7.80 (dd, J =
.8, 15.7 Hz, 4H), 7.73–7.68 (m, 2H), 7.53–7.48 (m, 4H), 7.32 (s, 2H),
.97 (s, 6H). 13C NMR (101 MHz, CDCl
8
8
3
1
1
3
) δ 156.61, 151.99, 148.18,
36.42, 129.88, 129.79, 129.10, 128.92, 127.61, 127.41, 126.79,
26.30, 118.81, 109.37, 77.42, 77.11, 76.79, 56.31. HR-MS (m/z, ESI)
Calculated for C30
H
24
2
N O
2
[M+H] m/z = 445.1911. Found m/z =
4
45.1902.
Quantum yield measure. The fluorescence quantum yields (Φ) of
DMQ in different solvents were determined by using fluorescein (0.1 M
NaOH, Φ = 0.95) as the standard reference, according to the literature
method [28]. The quantum yields were corrected as follows:
Scheme 1. The chemical structure of DMQ and its properties.
LDs with mitochondria and ER under the condition of starvation.
A
A
r
s
D
D
s
Φ
s
= Φ
r
r
where the s and r indices refer to designate the sample and reference,
respectively, A is the absorbance at λ ex, and D is the integrated area
under the spectrum.
2
. Experimental section
Cell culture and MTT assay. All cells were cultured in culture flasks
Regents and instruments. Glycerol trioleate (TG), glyceryl dioleate
in DMEM (high glucose) supplemented with fetal bovine serum (10%),
(
DG), cholesterol (CE), cholesteryl ester (CH), oleic acid (OA), p-dia-
◦
penicillin (100 U/mL) and streptomycin (50 U/mL) at 37 C in a CO
2
nisole, para-formaldehyde, formaldehyde solution, triphenylphosphine
and so on were purchased from Sigma. 3-(4,5-dimethythiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT), BODIPY 493/503, ER-tracker Red
and Mito-tracker DeepRed were purchased from Thermofisher. Other
chemical reagents were obtained from Sinopharm Chemical Reagent
Co., Ltd. Water was doubly distilled and purified via a Milli-Q water
system (Millipore, USA). NMR spectra in solution were performed on
Bruker 400 MHz Ultrashield Spectrometer. High-resolution mass spectra
2
incubator (95% relative humidity, 5% CO ). Cells were seeded into 20
mm glass-bottomed dishes, and cultured for 24 h for fluorescent
imaging.
The cytotoxicities of DMQ was studied by MTT assay. Hep G2 and
5
HeLa cells (10 cell/mL, 10 mL) were dispersed with 96-well microtiter
◦
plates to a total volume of 100
in 5% CO
μ
L/well. Plates were maintained at 37 C
2
/95% air incubator for 24 h. The cells were incubated for 18 h
with different concentrations of DMQ (1, 2, 4, 6, 8 and 10
μ
M) in the
(
HR-MS) were obtained by using an LTQ Orbitrap XL hybrid FTMS by
◦
medium at 37 C. Then, 10
μ
L MTT (5 mg/mL) solution was added into
Thermo Fisher Scientific. UV–visible absorption spectra were measured
with a HITACHI UH5300 spectrometer. Fluorescent spectra were
recorded on HITACHI F-7000 fluorescence spectrophotometer. Fluo-
rescent images were acquired on laser confocal microscope (Leica TCS
SP8X), and 50% of laser power was applied.
each well and incubated for additional 4 h. After removal of supernatant
and addition of 100 L of DMSO into each well, the cells were shaken for
0 min, and the absorbance in each well was measured at 492 nm use of
μ
1
a microplate reader (Biotek, USA). The cell viability (%) was calculated
according to the equation: cell viability % = A/B × 100%, where A
represents the absorbance of each well treated with DMQ, and B rep-
resents that of the control wells.
Synthesis of compound DMQ. The compound a and b were synthe-
sized according to the reported method [26,27].
Synthesis of compound a. The p-dianisole (5.55 g, 40.2 mmol) and
para-formaldehyde (1.52 g) were added into a 500 mL round bottom
flask, followed by adding aqueous formaldehyde solution (30%, 12.5
mL), concentrated hydrochloric acid (140 mL) and 1,4-dioxane (32.0
mL). After refluxing for 6 h, the mixture was cooled and filtered to
obtain the white precipitation as the crude product. The obtained
3
. Results and discussion
′
Firstly, we designed a highly hydrophobic probe, 2,2’-((1E,1 E)-(2,5-
dimethoxy-1,4-phenylene)bis(ethene-2,1-diyl))diquinoline (DMQ). The
synthetic route was shown in Scheme 2, and the structure of the target
powder was dissolved into CH
2 2
Cl , and then MeOH (600 mL) was added,
probe DMQ with a large
π
-conjugation was fully confirmed by HR-MS,
H and C NMR (Fig. S1-S3). In the structure of DMQ, there was no
strong hydrophilic moiety such as hydroxyl and amino groups (Scheme
). We further measured the oil-water separation coefficient (log P) of
the white precipitation which was collected by filtration and air dried to
1
13
1
afford compound a (7.18 g, yield 76%). H NMR (400 MHz, CDCl
.93 (s, 2H), 4.64 (s, 4H), 3.86 (s, 6H). HR-MS (m/z, ESI) Calculated for
12Cl
[M+H] m/z = 235.0287. Found m/z = 235.0276.
Synthesis of compound b. Compound a (2.6 g, 11.1 mmol), triphe-
3
) δ
6
1
C
10
H
2 2
O
DMQ as 3.034 (Fig. S4), suggesting our probe DMQ with high hydro-
phobicity. Then, the optical properties of DMQ in different solvents were
tested (Fig. 1). In the absorption spectra, DMQ in PBS buffer (pH 7.2)
showed a red-shifted peak and a broader band (Fig. 1a), likely because of
the aggregation formation of DMQ in aqueous solution (Fig. S9).
Meanwhile, DMQ had obvious fluorescence in different organic solvents
but no emission in PBS buffer (Fig. 1b and Table 1), indicating that the
aggregation of DMQ caused fluorescence quenching as a consequence of
the formation of an exciplex (Fig. S9) [29,30]. Notably, DMQ had strong
emission in TG, the major component of LDs core (Fig. 2a), which was
the basis of label intracellular LDs. To ensure the low noise in the next
imaging experiments, we further examined the DMQ fluorescent spectra
in liposomes, similar to the membrane structures in live cells. The results
indicated that no fluorescent signal was detected, which was like in PBS
buffer. These results imply that our probe DMQ is a candidate for label
LDs with high signal/noise ratio.
nylphosphine (8.8 g, 33.4 mmol) and methanol (70 mL) were added into
a 250 mL round bottom flask, and then the mixture was refluxed for 8 h.
After cooling down and removing the solvent in vacuum, a yellow oil
was obtained. Toluene (200 mL) was added to the foregoing mixture and
the generated white precipitation was filtered as the compound b (6.1 g,
1
yield 72%). H NMR (400 MHz, CDCl
3
) δ 7.79–7.61 (m, 30H), 6.92 (s,
2
H), 5.25 (d, J = 12.9 Hz, 4H), 2.96 (s, 6H). HR-MS (m/z, ESI) Calcu-
2
+
lated for C46
H
42
O
2
P
2
[M] m/z = 344.1325. Found m/z = 344.1318.
Synthesis of compound DMQ. Firstly, Na (0.12 g, 5.22 mmol) was
added to EtOH (14 mL) and stirred thoroughly until it was completely
dissolved to obtain a solution of sodium ethoxide. Quinoline-2-
formaldehyde (0.23 g, 1.46 mmol) and compound b (0.50 g, 0.66
mmol) were added into the above ethanolic solution of sodium ethoxide.
After refluxed for 4 h, the solvent was removed by rotary distillation
under vacuum. The solid was sequentially washed with n-hexane and
2

W. Ren et al.
Dyes and Pigments 191 (2021) 109366
Scheme 2. The synthetic route of probe DMQ.
Fig. 1. (a) UV–vis spectra of DMQ in different solvents. (b) Fluorescent spectra of DMQ in different solvents.
the almost non-cytotoxicity of probe.
Table 1
For cell imaging, we set the imaging channel as excitation at 405 nm
Summary of the absorbance maximum, emission peaks, Stokes’ shift, the
quantum yields of DMQ in different solvents.
and emission collection in 450–550 nm. After the cells were incubated
with 1 M DMQ, the dot-like bright fluorescence was observed (Fig. 3a),
μ
Solvent
Absorbance
nm)
Emission
(nm)
Stokes’ shift
Φ
and there was almost no noisy signal. To confirm that the fluorescent
dots were LDs, the available commercial LD-tracker, BODIPY 493/503,
was employed for the colocalization experiment (Fig. 3b and c). When
(
(nm)
Petroleum ether
Cyclohexane
Chloroform
Ethyl acetate
Acetone
408.0
410.0
418.0
414.0
414.0
414.0
424.0
456.0
457.6
476.0
473.0
478.6
488.6
493.6
48.0
47.6
58.0
59.0
64.6
74.6
69.6
0.729
0.642
0.440
0.494
0.494
0.440
0.385
the DMQ-loaded cells were further treated with 1 M BODIPY 493/503,
μ
it was found that the fluorescent dots almost completely overlapped,
suggesting the probe DMQ accumulated in intracellular LDs. The same
results were obtained in other two cell lines (Fig. S8). It is worthy
pointing out that the noise in Fig. 3b showed much stronger (Fig. 3d) but
weaker LDs signal detected. The similar results were also observed by
using Nile Red (Fig. 3e and j), suggesting that our probe DMQ had a
higher signal/noise ratio due to the negligible fluorescence of probe in
aggregation state and in liposomes. These results indicate the probe
DMQ more suitable to investigate interactions between LDs and other
organelles.
Methanol
Dimethyl
sulfoxide
PBS
436.0
608.6
172.6
0.004
Next, we tested the fluorescence stability in the complicated bio-
environments, because there are various reactive bio-species may
interfere the fluorescence of DMQ. We added common biomolecules
(
including inorganic salts, amino acids, peptides, proteins, and nucleic
Next, we tried to use our probe to track LDs growth when the cells
were treated with supplementary oleic acid (Fig. 4a). OA is an organic
acid, and thus can be ionize partly in water and taken by cells. We have
calculated the mean size of LDs, and the results were shown in Fig. 4b
and c. It was found that the numbers and sizes of LDs exhibited the
acids) into DMQ aqueous solution, and found no obvious fluorescent
change (Fig. S6). In parallel, we also investigated the fluorescence of
DMQ in the presence of other components in LDs such as dioleate
glyceryl (DG), oleic acid (OA), palmitic acid (PA), cholesterol (CE) and
cholesterol ester (CH) (Fig. 2b), and the fluorescence of DMQ kept
relatively steady. The above results showed that intracellular bio-
molecules cannot interfere the DMQ emission, avoiding the generation
of noise from biomolecules. Before moving to cellular imaging, the
cytotoxicity of probe was measured by the classic MTT method (Fig. S7).
similar trend in the presence of oleic acid. In the initial stage, the LDs
2
were relatively small with a mean area of 0.04
μ
m . In 0–2 h, the fluo-
rescence intensity and size of LDs gradually increased under the treat-
ment of oleic acid. Because DMQ with high hydrophobicity can easily
accumulate into intracellular LDs, and the high concentration of DMQ in
LDs may induces aggregation caused quenching (ACQ). With the uptake
The cell viability kept over 90% in the presence of 1–10
μM, indicating
3

W. Ren et al.
Dyes and Pigments 191 (2021) 109366
Fig. 2. (a) UV–Vis absorption and fluorescence spectra of DMQ (10
fluorescence intensities at the emission peak (490 nm) of DMQ in different lipids environments. The different lipids environments contained 90% of TG and 10%
other lipids, dioleate glyceryl (DG), oleic acid (OA), palmitic acid (PA), cholesterol (CE) and cholesterol ester (CH). The error bars represent mean errors from the
results of three tests.
μ
M) in TG with the excitation of 405 nm, and collected in the range of 450–550 nm. (b) The
Fig. 3. Fluorescent images and profiles of Hep G2 cells stained with DMQ (1
μ
M) and BODIPY 493/503 or Nile Red (1
μ
M). The cells were incubated with DMQ for
2
0 min, and then treated with BODIPY 493/593 or Nile Red for another 20 min. (a) The image from DMQ channel. (b) The image from BODIPY 493/503 channel. (c)
Overlay of (a) and (b). (d) Fluorescence intensity profile of region of interest (blue line in a and b). (e) Signal/noise ratios in (a) and (b). (f) The image from DMQ
channel. (g) The image from Nile Red channel. (h) Overlay of (f) and (g). (i) Fluorescence intensity profile of region of interest (blue line in f and g). (j) Signal/noise
ratios in (f) and (g). DMQ, BODIPY 493/503 and Nile Red were excited with 405, 493 and 550 nm, and collected in the range of 450–550, 500–550, and 600–650 nm,
respectively. The error bars represent mean errors from the results of three tests.
of oleic acid, LDs’ volume became bigger than before, resulting in the
decrease of DMQ concentration and the increase of fluorescence in-
tensity. In the period of 2–4 h, both the numbers and areas of LDs
increased sharply, while the increase speed obviously decelerated after
[31]. To demonstrate these physiological behaviors, we employed the
probe DMQ to study the interactions of LDs with mitochondria and ER.
When we stained the cells with DMQ and Mito-tracker simultaneously,
the LDs had no obvious communication with mitochondria, and their
overlap coefficient as low as 0.03 (Fig. 5a). However, after the further
incubation in nutrition-free media for 10 min, the overlap coefficient
increased to 0.06, and rapidly enhanced 6-fold in another 1 h. In the
control group of high glucose media, the overlap of LDs with mito-
chondria still kept in very low level (Fig. 5b and c). The results suggest
that the more contacts occurred between LDs and mitochondria. In the
case of DMQ and ER tracker, the similar results were also observed.
Under the starvation condition, the overlap coefficient between LDs and
ER also increased from 0.20 to 0.33, whereas the control group kept
relative steady (Fig. 6). To confirm the results, we have using the
4
h. The results showed that the sizes and numbers of LDs both increased
upon the uptake of oleic acid (Fig. 4b and c), indicating that our probe
DMQ was suitable to real-time monitor LD’s growth in live cells. Inter-
estingly, there was no detectable fluorescent signal in ER and other
membrane-structural organelles, which further certifying that the probe
DMQ can also be applied in the investigation of LDs contacting other
organelles.
As well-documented, LDs as the source of intracellular lipids are
often linked to other organelles, such as providing fatty acids to mito-
chondria and supplying lipid materials to ER in starvation condition
4

W. Ren et al.
Dyes and Pigments 191 (2021) 109366
Fig. 4. (a) Fluorescence imaging of HeLa cells. The cells were first incubated with probe DMQ (1
μ
M), and then directly treated with 100 M oleic acid (OA) for
μ
different times as indicated in the corresponding images. (b) Statics numbers and areas of LDs at different times. (c) Statics average diameter of LDs at different times.
Here, DMQ were excited with 405 nm, and collected in the range of 450–550 nm. Error bars represent mean errors from the results of three tests.
Fig. 5. Fluorescent imaging of the interactions between LDs and mitochondria. (a) Imaging of cells in starvation conditions. The cells were simultaneously incubated
with DMQ (1
cells were simultaneously incubated with DMQ (1
mented with 10% FBS. (c) The time-dependent Pearson’s correlation of lipid droplets and mitochondria. Here, DMQ and Mito-tracker Deep Red were excited with
05 and 633 nm, and collected in the ranges of 450–550 and 645–665 nm, respectively. The error bars represent mean errors from the results of three tests.
μ
M) and Mito-tracker Deep Red (1
μ
M) for 20 min, then cultured in 1.0 mL of serum-free Earle’s balanced salt solution. (b) Imaging of control cells. The
μ
M) and Mito-tracker Deep Red (1 M) for 20 min, then cultured in 1.0 mL of a nutrient-rich medium supple-
μ
4
Fig. 6. Fluorescent imaging of the interactions between LDs and ER. (a) Imaging of cells in starvation conditions. The cells were simultaneously incubated with DMQ
M) and ER-tracker Red (1
M) for 20 min, then cultured in 1.0 mL of serum-free Earle’s balanced salt solution. (b) Imaging of control cells. The cells were
simultaneously incubated with DMQ (1 M) and ER-tracker Red (1 M) for 20 min, then cultured in 1.0 mL of a nutrient-rich medium supplemented with 10% FBS.
(
1
μ
μ
μ
μ
(
c) The time-dependent Pearson’s correlation of lipid droplets and ER. Here, DMQ and ER-tracker Red were excited with 405 and 587 nm, and collected in the ranges
of 450–550 and 600–650 nm, respectively. The error bars represent mean errors from the results of three tests.
5

W. Ren et al.
Dyes and Pigments 191 (2021) 109366
commercial LDs dyes to carry out these experiments (Fig. S10 and S11),
and the performance of commercial LDs dyes had the similar trends to
that using our probe DMQ, but the overlaps of LDs and other organelles
in the initial stage was higher, leading to the decrease of sensitivity. In
particular, using BODIPY 493/503 (one of the commercial LDs dyes) had
no enhancement in 0–20 min, which was contradictory to the fact.
Together all, our probe with high signal/noise ratio was suitable to
real-time monitor the interactions of LDs with other organelles in live
cells.
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Wei Ren: Methodology, Data curation; Dong Wang: Data curation;
Wei Huang: Data curation; Jiajia Li: Data curation; Xiaohe Tian:
Writing-Original draft; Zhengjie Liu: Cell culture; Guangmei Han:
Imaging; Ruilong Zhang: Funding, Supervision, and Writing; Bianhua
Liu: PI, Supervision, Funding, and Reviewing; Ming-Yong Han: PI,
Supervision, Funding, and Reviewing; Zhongping Zhang: Funding,
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