A Fluorescent Chemodosimeter for Organelle-Specific Imaging of Nucleoside Polyphosphate Dynamics in Living Cells

Article abstract of DOI:10.1021/acs.cgd.8b01409

Nucleoside polyphosphates (NPPs) are mainly produced in mitochondria and used as a universal energy source for various cellular events. Although numerous fluorescent probes for adenosine triphosphate (ATP) have been reported, they are not ideally suited for live monitoring of the subtle variation of the mitochondrial ATP level. A new coumarin-based fluorescent probe is synthesized, and this reagent is utilized for specific recognition of NPPs in mitochondria by super-resolution microscopy in physiological condition. Detailed ³¹P NMR studies reveal that the probe, L·Zn(II), binds to NPPs through their pendent phosphate functionalities. Such binding leads to a substantial enhancement in the luminescence intensity of L·Zn(II) + ADP or L·Zn(II) + ATP as compared to L·Zn(II). This - as well as ¹H NMR spectroscopy - has enabled us to evaluate the probe's binding affinities to these NPPs. Structured illumination and wide field fluorescence microscopy confirmed that this physiologically benign reagent is localized within mitochondria of live RAW 264.7 macrophage cells. This reagent has been utilized to probe real-time changes in ATP concentrations within mitochondria during drug-induced apoptosis.

Full text of DOI:10.1021/acs.cgd.8b01409

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Article
A Fluorescent Chemodosimeter for Organelle-Specific Imaging
of Nucleoside Polyphosphate Dynamics in Living Cells
Harwinder Singh, Sreejesh Sreedharan, Rajeshwari Tiwari, Christa. Walther,
Carl G W Smythe, Sumit Pramanik, Jim Antony Thomas, and Amitava Das
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01409 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 11, 2018
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A Fluorescent Chemodosimeter for Organelle-Specific Imaging of Nucleoside
Polyphosphate Dynamics in Living Cells
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Harwinder Singh, Sreejesh Sreedharan, Rajeswari Tiwari, Christa Walther, Carl Smythe,
Sumit Kumar Pramanik, Jim. A. Thomas and Amitava Das
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New fluorescence-based reagent for probing subtle changes of NPPs in mitochondria
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A Fluorescent Chemodosimeter for Organelle-
Specific Imaging of Nucleoside Polyphosphate
Dynamics in Living Cells
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Harwinder Singh,a Sreejesh Sreedharan,b,c Rajeswari Tiwari,a Christa Walther,d Carl Smythe,e
Sumit Kumar Pramanik,a* Jim. A. Thomasb* and Amitava Dasa*
a CSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar: 364002, Gujarat,
India; E-Mail: sumitpramanik@csmcri.res.in (SKP); a.das@csmcri.res.in (AD)
b Current address: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology,
University of Oxford, Oxford OX3 7DQ, UK
c Department of Chemistry, University of Sheffield, Sheffield, S3 7HF, UK, Email:
james.thomas@sheffield.ac.uk.
d Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S3 7HF, UK.
e Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, UK.
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ABSTRACT
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Nucleoside polyphosphates (NPPs) are mainly produced in mitochondria and used as a
universal energy source for various cellular events. Although numerous fluorescent probes for
Adenosine Tri-Phosphate (ATP) have been reported, they are not ideally suited for live
monitoring of the subtle variation of mitochondrial ATP level. A new coumarin-based
fluorescent probe is synthesized and this reagent is utilized for specific recognition of NPPs in
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mitochondria by super resolution microscopy in physiological condition. Detailed P NMR
studies reveal that the probe, LZn(II), binds to NPPs through their pendent phosphate
functionalities. Such binding leads to a substantial enhancement in the luminescence intensity
1
of LZn(II)+ADP or LZn(II)+ATP as compared to LZn(II). This - as well as H NMR
spectroscopy - has enabled us to evaluate the probe’s binding affinities to these
NPPs. Structured illumination and wide field fluorescence microscopy confirmed that this
physiologically benign reagent is localized within mitochondria of live RAW 264.7
macrophage cells. This reagent has been utilized to probe real-time changes in ATP
concentrations within mitochondria during drug-induced apoptosis.
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1. INTRODUCTION
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Nucleoside polyphosphates (NPPs) are considered to be the main energy vectors of the cell and
play pivotal roles in various cellular events.1-2 The most familiar NPP, Adenosine Tri-
Phosphate (ATP) is seen as the primary energy currency. It is largely produced in mitochondria
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and regulates cellular metabolism.3 ATP is an important marker for studying cell injury, tumor
3-4
progression/ cancers and many critical diseases.
In addition, NPPs are crucial for
intracellular and extracellular signaling process.2 In mammalian tissues, the release of NPPs
from synapses and axons activates purinergic receptors, which modulate intracellular calcium
and cyclic AMP levels.5-6 In the central nervous system, ATP modulates neural development,
the control of immune systems, and neuron/glial signaling.5, 7 NPPs are also involved in signal
transduction as their phosphate groups are used as substrates for kinases in phosphate transfer
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reactions which activate a cascade of protein kinase reactions.
Besides these intracellular
10-12
roles, NPPs are extracellularly controlled by various chemical and mechanical stimuli.
Thus, intracellular ATP concentrations quickly vary in response to a wide variety of external
stimuli, including cytotoxic agents, nutrients, hypoxia, and hormones.
In eukaryotic cells, ATP is mostly synthesized during glucose metabolism in mitochondria
through oxidative phosphorylation. 13-14 During the transfer of hydrogen atoms from FMNH2
or FADH2 to oxygen, protons are pumped across the mitochondrial membrane from the inside
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of the mitochondrion to the outside.
This creates an electrical potential across the
mitochondrial membrane, corresponding to 200-300 millivolts, and it is this electrical energy
that is converted into chemical energy by ATP synthase enzyme, generating ATP from ADP.
17 Alterations of mitochondrial membrane potential in eukaryotic cells leads to mitochondrial
dysfunction and this appears as a common phenotype in mitochondrial diseases affecting ATP
production. 17
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Therefore, methods to specifically detect and quantify ATP are extremely important. It has
been argued that subtle variations in the concentration of NPPs within mitochondria are also
indicative of various disease conditions. 3-4, 18 Thus, a highly sensitive, specific and quantitative
detection platform for the real-time monitoring of NPPs is a highly attractive goal within the
context of clinical diagnostic. Whilst, various detection methods for NPPs have been reported,
19-23 fluorescent probe that localizes in the mitochondrion are highly suited for this application,
as such reagents allows selective monitoring of biological events as they occur inside this
organelle.18, 24-27 Hence, the synthesis of organelle-specific molecular probes that produce a
luminescence ON response on binding to, or reacting with, NPPs would have significance for
clinical diagnosis, cell biology and assessing analytical samples. Yet contemporary literature
reports on fluorescent chemodosimeters that are proficient in analyzing the spatial and temporal
dynamics of NPPs in mitochondria are rare.
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Conventional fluorescence microscopy allows non-invasive imaging of lysosomes and
mitochondria in live cells.28 However, it suffers from an optical diffraction-limited spatial
resolution and fails in resolving the membrane boundaries between lysosomes and
mitochondria.28 Although electron microscopy can overcome this limitation, it fails in
capturing dynamic processes in live cells. More recently many of the drawbacks of
conventional imaging modalities have been addressed through super-resolution microscopy
(SRM) techniques, which can overcome the diffraction limit of conventional optical but can
still be employed to monitor dynamic processes. One such SRM technique is SIM (structured
illumination microscopy), which provides an appreciable increase in resolutions (to 100–120
29-30
nm) with negligible disruption of data acquisition rate.
SIM requires considerably lower
illumination intensities compared to other super-resolution microscopies like STORM and
30-32
STED, making it particularly suited for live cell imaging.
mitochondrial probes undergo photo-bleaching under SIM.33
However, most existing
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In this manuscript, we report a turn-on luminescent probe for NPPs that spontaneously localizes
in mitochondria, without anchor-protein expression. Taking advantage of its ability for
selective subcellular internalization, this chemodosimetric probe has been successfully used to
image the subtle changes in the effective concentration of NPPs within living cells at super-
resolutions. This has allowed us to detect the extracellular release of NPP from living cells and
observe stimuli-responsive change in ATP concentration in mitochondria. To the best of our
knowledge, this probe provides unique capabilities for the visualization of NPPs dynamics in
the mitochondria of live cells, along with simultaneous detection of changes in ATP
concentration within cellular compartments using multicolor imaging.
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2. EXPERIMENTS AND METHODS
Materials.
All common chemicals and solvents were procured from commercial suppliers and used
without any further purification unless mentioned otherwise. 7-Diethylamino-4-
methylcoumarin (99.0%), dichloromethane (DCM) (> 99.0%), Selenium dioxide (> 99.0%),
Methanesulfonyl chloride (99%), Sodium borohydride (98%), Zinc perchlorate hexahydrate,
Di-(2-picolyl)amine (DPA), Xylene (> 99.0%), Lithium bromide (> 99.0%), phosphate buffer
saline (PBS), fetal bovine serum, penicillin−streptomycin, 4% paraformaldehyde (PFA),
Vectashield h-1000 (mounting agent), 50 mM ammonium chloride, Lyso tracker red, Mito
tracker deep red, and Hoechst (33342) were purchased from Sigma-Aldrich. Iscove’s Modified
Dulbecco’s Medium (IMDM), fetal calf serum (FCS), Dulbecco’s Modified Eagle’s medium
(DMEM), Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) antibody, mouse monoclonal anti-
α-tubulin antibody, and 4′,6-Diamidino-2-Phenylindole (DAPI) were purchased from Life
Technologies. The 15 μ-slide 8 wells were purchased at Ibidi. The culture plates and dark plates
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were obtained from Greiner Bio One. RAW 264.7 macrophage cell line was purchased from
ATCC.
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Synthesis of the probe LZn(II).
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To a solution of 3 (0.277 g, 1.12 mmol) in anhydrous CH2Cl2, methanesulfonyl chloride (0.13
mL, 1.68 mmol) was added at 0° C. After 3 was consumed (as determined by TLC), saturated
NaHCO3 solution (20 mL) was added. The aqueous phase was extracted with CH2Cl2 (3 × 50
mL) and the combined organic phases were dried and concentrated. The obtained crude product
was immediately dissolved in anhydrous THF (10 mL). Anhydrous lithium bromide (0.292 g,
3.36 mmol) was added to the solution and the mixture was stirred at room temperature for 1
hour. The solvent was evaporated and the crude product was purified by column
chromatography (PE/EA = 2.5:1) to afford product 4 as orange crystals (0.285 g, Yield: 82 %).
1H NMR (600 MHz, CDCl3): δ 1.18 (6H, t, J = 6.0 Hz), 3.39 (4H, q, J = 6.0 Hz), 4.37 (2H,
s); 6.10 (1H, s); 6.48 (1H, s), 6.59 (1H, d, J = 12.0 Hz); 7.46 (1H, d, J = 12.0 Hz). 13C NMR
(150 MHz, CDCl3): δ 12.43, 27.08, 44.86, 97.86, 106.04, 108.75, 109.14, 125.29, 150.23,
151.03, 156.73, 161.77.
To a round-bottom flask equipped with a stir bar was charged with 4 (0.168 g, 0.56 mmol), Di-
(2-picolyl)amine (0.1 mL, 0.56 mmol) and Na2CO3 (0.350 g, 3.33 mmol). Then, CH2Cl2 (20
mL) was added. The reaction mixture was stirred at 140 oC for 24 h until purine was consumed
(determined by TLC). Then, the solvent was removed and purified by column chromatography
on silica gel (PE/ 85% EtOAc followed by CH2Cl2/5% MeOH) to afford L as light yellow solid
(0.211 g, Yield: 91 %).
1H NMR (600 MHz, CDCl3): δ 1.20 (6H, t, J = 6.0 Hz); 3.4 (4H, q, J = 6.0 Hz); 3.82 (2H, s);
3.90 (2H, s); 6.48 (1H, s), 6.49 (1H, d, J = 6.0 Hz), 6.54 (1H, d, J = 12.0 Hz); 7.18 (2H, t, J =
6.0 Hz); 7.47 (1H, d, J = 12.0 Hz); 7.54 (2H, d, J = 6.0 Hz) 7.68 (2H, t, J = 6.0 Hz), 8.54 (2H,
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d, J = 6.0 Hz). 13C NMR (150 MHz, CDCl3): δ 12.41, 44.90, 54.25, 60.74, 97.83, 107.79,
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108.16, 122.30, 123.06, 125.26, 136.77, 148.99, 150.32, 153.75, 156.33, 158.51, 162.48.
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A round-bottom flask equipped with a stir bar was charged with 1 (1.0 g, 4.32 mmol), and SeO2
(0.958 g, 8.64 mmol). Then, xylene (20 mL) was added. The reaction mixture was stirred at
room temperature for 24 h until 1 was completely consumed (determined by TLC). Then the
reaction was filtered and the organic phase was concentrated. The obtained crude product (2)
was immediately dissolved in ethanol (200 mL) and anhydrous sodium borohydride (0.49 g,
12.96 mmol) was added and the mixture was stirred at room temperature for 12 hour. Then,
the solvent was removed and purified by column chromatography on silica gel (PE: EtOAc is
1:1) to afford 3 as yellow solid (0.765 g, Yield: 72 %).
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1H NMR (600 MHz, CDCl3): δ 1.19 (6H, t, J = 6.0 Hz), 3.39 (4H, q, J = 6.0 Hz), 4.82 (2H,
s); 6.27 (1H, s); 6.47 (1H, s); 6.55 (1H, d, J = 12.0 Hz); 7.30 (1H, d, J = 6.0 Hz). 13C NMR
(150 MHz, CDCl3): δ 12.29, 44.62, 60.48, 97.62, 105.21, 106.22, 108.81, 124.48, 150.20,
155.23, 156.15, 163.03.
To a solution of L (0.500 g, 1.03 mmol) in MeOH, Zn(ClO4)2, 6H2O (0.521 g, 1.4 mmol, in 10
mL HPLC water) was added in a drop wise manner. The resultant solution mixture was allowed
to stir for 6 h at 20 oC. A yellow coloured precipitate appeared, which was filtered off and dried
in air to obtain the L•Zn(II) complex in a pure form (0.523 g. Yield: 90%).
1H NMR (600 MHz, DMSO-d6): δ 8.45(2H, d, J= 4.0 Hz), 7.77 (2H, t, J = 4.0Hz), 7,58 (2H,
d, J = 4.0), 7.54 (1H, d, J = 8.0 Hz), 7.27 (2H, t, J = 4.0 Hz), 6.64 (1H, d, J = 8.0 Hz), 6.42
(1H, s), 6.29 (1H, s), 3.89 (4H, s), 3.84 (2H, s), 3.43 (4H, d, J = 4.0 Hz), 1.17 (6H, t, J = 4.0
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Hz). C NMR (CDCl3, 125 MHz): 160.92, 155.86, 154.61, 151.14, 148.04, 141.19, 126.23,
125.37, 111.36, 109.25, 108.42, 97.69, 57.00, 44.43, 12.91.
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Cell Culture
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RAW 264.7 macrophage cell line were incubated in DMEM supplemented with 10% (v/v)
fetal bovine serum and 1% (w/v) penicillin-streptomycin at 37 °C in presence of 5% of CO2
in the humidified air. Cells were passaged when they are approximately 80% confluence.
Then the cells were seeded onto a cell culture dish at a density of 1.0 × 105 cells incubated at
37 °C under 5% CO2 in the humidified air.
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MTT Assay
Initially, the molecular probe LZn(II) was tested for cytotoxicity towards RAW 264.7
macrophage cell line. RAW 264.7 macrophage cell were placed in a 96 well plated at a density
of 1.0 x 105 cells/well in DMEM medium (Gibco), supplemented with 5% fetal bovine serum
along with 100 units of penicillin−streptomycin antibiotics. The cells were incubated at 37 °C
in a 5 % CO2 incubator for 24 h after which they were washed with 1 x PBS. A total of six
wells per condition were taken and 100 µL were added to the respective wells: blank
(supplemented IMDM medium), cell death, 120 µM, 60 µM, 30 µM and 15 µM of probe
molecule LZn(II) in the supplemented IMDM medium. The cells were left to incubate for 48
h in the incubator after that these cells were washed thrice with 1 x PBS. Then 100 µL of 10%
MTT
[3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium
bromide]
solution,
in
supplemented IMDM medium, was added to each well and further incubated for another 4 h.
Then the solution was transferred to a dark plate and the absorbance measured at 570 nm. Each
experiment was performed in triplicate.
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Microscopy Experiments
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Deconvolution Widefield Microscopy
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To remove the out of focus blur from stacks of acquired Z-Stack images, deconvolution of raw
widefield images acquired using the OMX-SIM (Conventional Wide Field Microscopy mode)
was carried out using the Soft Worx software.
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Cellular uptake of LZn(II) complex (Single colour Imaging)
Single colour widefield experiments using LZn(II) were performed at a range of
concentrations (10 μM to 200 μM) of LZn(II) and uptake studies were carried out over 24
hours. For single colour widefield experiments, an excitation wavelength of 400 nm was used
and luminescence was collected at 500 to 550 nm. The widefield microscopy conditions
maintained were; Z stack thickness (Sections 40 to 80), section spacing (0.250 to 0.500), and
thickness of the sample (8 to 11). Selected exposure times were between 10 to 30 s and the %T
was in the range of 30 to 60.
Colocalization Experiments (Multicolour imaging)
Individual colocalization experiments were performed using LysoTracker Deep Red or Mito
Tracker Deep Red. For these colocalization experiments, the RAW 264.7 macrophage cell line
were incubated with 50 μM of LZn(II) for 4 hours and then the cells were incubated with LTDR
or MTDR (750 nM) for 30 minutes. These pre-treated cells (fixed with 4% PFA and mounted)
were washed thrice with DMEM culture media and then thrice with PBS. The LZn(II)
complex was excited at 400 nm and the emission was collected in the FITC Channel (500 to
550 nm). For these studies, Lyso or Mito Tracker Deep Red or Hoechst was excited at 644 nm
and emission was collected in the Alexa Fluor 647 Channel (> 650 nm).
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SIM Microscopy - Single colour, Colocalisation, and Dual colour experiments
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Structured illumination microscopy (SIM) was carried out by using the Delta Vision OMX-
SIM. The Z stacks acquired during the imaging were post-processed by using the
reconstruction option of Soft Worx. For single colour experiments, the LZn(II) complex was
excited at 400 nm and the emission was collected at FITC Channel (500 to 550 nm). The
Structured Illumination (SI) experimental condition employed for running the SI experiment
for single colour experiments were mainly dependent on the thickness of the Z stack (Sections
80 to 100), section spacing (0.125 to 0.250), the thickness of the sample (8 to 10). The dual
colour experiments were performed in combination with Hoechst.
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3. RESULTS AND DISCUSSION
We chose coumarin as a luminescence-based reporter functionality due to its excellent
photophysical properties, such as high molar absorptivity, excellent photostability, tuneable
emission through appropriate derivatization, as well as a relatively narrow emission bandwidth
with high luminescence quantum yield. 34-35 The synthesis of LZn(II) complex is outlined in
Scheme 1. All the characterization data are provided in the supporting information.
Scheme 1 The synthetic route towards L•Zn(II)complex.
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The absorption spectrum of LZn(II) was recorded in an aqueous HEPES buffer solution at pH
7.4. A strong absorption band with a maximum at 400 nm (ε = 38000 L mol-1 cm-1) was
observed and this was attributed to an S0–S1 intramolecular charge transfer (ICT) with the
amine functionality as the donor and the coumarin moiety as the acceptor fragment. During
titration with Zn(ClO4)2, the luminescence intensity (Ext = 400 nm) of ligand L was found to
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LZn II


decrease slowly and eventually a weak emission band (
= 0.0136) with a maximum at
500nm
500 nm was observed upon addition of 2 mole equiv. of Zn(ClO4)2 (Figure S1). Binding of the
Zn(II)-centre to the bis picolyl moiety (Scheme 1) would further favor the ICT process and
account for a narrower HOMO–LUMO energy gap. The smaller energy gap between frontier
orbitals promotes non-radiative deactivation of the excited state and hence, the lowered
emission quantum yield of LZn(II). The luminescence spectral response of LZn(II) towards
a variety of anionic analytes was tested using 50-mole equivalents of the respective analytes.
No noticeable change in luminescence profile was observed when spectra were recorded in the
2
presence of all other analyte (e.g., X: H2O2, OHˉ, HNO, Na2S2, NO3ˉ, NO2ˉ, NO3ˉ, SO4 ˉ, SO3ˉ,
S2O3ˉ, Clˉ, urea, PPi, AMP, creatinine and all natural amino acids (AA)), except for NPPs
(ADP and ATP) (Figure S2). On treatment with NPPs, a distinct enhancement in luminescence
LZn II ATP


intensity was observed for the luminescence band centered at 500 nm (
= 0.341 and
500nm
LZn II ADP
500nm



= 0.337 for Ex = 400 nm; coumarin was used as a standard to estimate relative
emission quantum yields).
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These observations are consistent with other reports showing that Zn(II) systems have an
affinity towards ATP and ADP; 22, 36 for example, the Zn(II)-centre in Zn(DPA) or Zn(cyclam)
bind to ATP and/or ADP through their pendent phosphate moieties (Figure 1). 21, 37-40
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


L.Zn(II) + ATP
N-Zn(II) bond
Distance (L1 Å)



N-Zn(II) bond
Distance (L2 Å)
ATP
L2 >> L1: Much longer N-Zn(II) bond distance in LZn(II)ATP would weaken the binding
between the DPA moiety and Zn(II) with subsequent weakening of the ICT process,
which was otherwise operational in LZn.
 (ppm)
Figure 1. (a) Molecular structure showing the probable binding of ATP and ADP to LZn(II) and (b) partial 31
P
NMR spectra of ATP and ATP bound to LZn(II).
This possibility for LZn(II). was confirmed from the results of the P31 NMR studies (vide
infra). This binding is expected to decrease the interaction between the DPA moiety and Zn(II)
and subsequent weaken the ICT process, which is otherwise operational in the complex. This
effect contributes to the observed enhanced luminescence intensity.
1.0x105
8.0x104
6.0x104
4.0x104
2.0x104
0.0
0.0
9.0x10-5
[ATP] (M)
1.8x10-4
Figure 2. A plot of change in luminescence intensity at 500 nm recorded for a solution of L•Zn(II) (1.0 μM) with
subsequent changes in [ATP] (0 – 10 M) in 50 mM of aq. HEPES buffer solution having10 mM NaCl, 1 mM
MgCl2 (pH = 7.4, 25 °C, λExt = 400 nm).
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Binding of ATP to the Zn(II)-centre of LZn(II) was further confirmed by 31P NMR spectral
studies. Upfield shifts in the 31P signals of the α- (1.84 ppm), β- (3.92 ppm) and γ- (2.39 ppm)
of the phosphorus atoms of ATP bound to LZn(II) were observed (Figure 1). These shifts
signify the binding to Zn-atom of LZn(II) through an oxygen atom containing the negative
charge of the three conjugated phosphate units. This lead to the formation of a hepta-
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coordinated Zn(II)-centre in LZn(II) in presence of ATP (Scheme 2). Analogous P NMR
studies with ADP, showed shifts of 2.09 and 1.81 ppm for two P-atoms of ADP and this
confirmed formation of a penta-coordinated Zn(II)-centre in LZn(II) (Figure S3).
The probe molecule LZn(II) shows a relatively large Stokes shift of ~100 nm, a highly
desirable property for improved luminescence responses as it helps to reduce self-absorption
and background noise as it leads to less interference from endogenous fluorophores. Systematic
changes in the luminescence spectra as a function of varying [ATP] and [ADP] (0 to 100-mole
equivalent) in HEPES buffer were recorded (Figures 2 (for ATP) and Figure S4 (for ADP)). A
linear increase in luminescence intensity was observed until the [ATP] or [ADP] was 5 μM and
a lower detection limit was evaluated as 8.4 x 10-9 MM for ATP and 1.25 x 10-8 M for ADP,
respectively using the 3σ method (Figure S5). This also confirmed the high sensitivity of the
chemodosimetric probe LZn(II) towards these NPPs.
The luminescence responses of LZn(II) (10 μM) were recorded after incubating with 100 mole
equivalent of ATP/ADP (recorded after 20 min) in the absence and presence of large excess
(1000 mole equiv.) of other anionic analytes / amino acids/ biomolecules (Figure S6). No
further change in luminescence intensity was observed when spectra were recorded in the
presence of all other possible competing anions/amino acids/ biomolecules. This clearly
nullified any interference with other competing amino acids and biomolecules and confirmed
the desired specificity of LZn(II) towards ATP/ ADP.
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A short response time is also a very important criterion for developing an analytical reagent for
quantitative estimation of biomarkers for any practical application.41-42 To determine the
response time of the probe LZn(II) towards ATP/ADP, luminescence intensities as a function
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of time in the absence and presence of ATP or ADP were monitored at 500 nm (Ext = 400
nm). Although no detectable change in luminescence intensity with time was observed in the
absence of ATP/ADP (Figure S7), the increase in luminescence intensity on addition of ATP
or ADP, took ~3.8 min to plateau.
MTT assays were performed to check the cytotoxicity of the reagent towards RAW 264.7
macrophage cells. No decrease below 99% in cell viability was observed even after exposures
to all tested concentrations of LZn(II) for 24 h (Figure S8). This confirmed the
biocompatibility and the benign nature of the reagent LZn(II) towards live cells like RAW
264.7 macrophage cells. Thus, the reagent LZn(II) was ideally suited for application as an
imaging reagent.
Widefield fluorescence microscopic images of RAW 264.7 macrophage cells after incubation
with the reagent LZn(II) revealed that intracellular luminescence was almost entirely localized
in the cytoplasmic region (Figure 3). Additionally, increases in [LZn(II)] resulted in a
concomitant increase in intracellular emission. This was also evident in intensity maps
generated from these data (Figure S9), which revealed the punctated nature of the images,
indicating that LZn(II) was localized in specific regions of the cytoplasm. This was
investigated in detail through co-localization studies using SIM.
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(a)
(c)
(b)
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Figure 3. Confocal microscopy images of intracellular uptake of L•Zn(II) using Hoechst33342: confocal
microscopy images of in cellulo emission of L•Zn(II) (panel a) with expansion shown underneath. Emission from
Hoechst33342 (panel b) and the expansion of the same is shown below. The overlap of the intensity is shown in
panel c and with expansion shown underneath. panel c shows the overlap of the green and red fluorescence,
indicating intracellular green luminescence was almost entirely localized in the cytoplasmic region. Ext = 400
nm.
(a)
(b)
(c)
(e)
2 μm
2 μm
2 μm
Mito Peak
LZn(II)
(d)
2.0x104
1.5x104
1.0x104
5.0x103
1.0x104
8.0x103
6.0x103
4.0x103
2.0x103
Pearson’s
Coeff: 0.938
0
5
10
15
20
0
5
10
15
20
Distance (m)
Distance (m)
2 μm
Figure 4. Colocalization experiments of intracellular localization of LZn(II) using MitoTracker probes:
Widefield microscopy images of in cellular emission of LZn(II) (panel a) with intensity along the traced line
shown underneath. Emission from Mito Tracker Deep Red (panel b) and intensity along the same line shown
below. The overlap of the intensity is shown in panel c and its expansion in panel d. Panel c shows the overlap of
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the green and red fluorescence, indicating mitochondria localization of LZn(II). Panel e shows the Pearson
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coefficient = 0.92. Scale bar 2 μm.
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It is reported that the negative membrane potential of the mitochondrial inner membrane favors
the accumulation of positively charged compounds in the mitochondrial matrix against their
concentration gradient. 27, 43 Thus, the cationic nature of probe LZn(II) is expected to favor its
accumulation in mitochondria.44 To ascertain this hypothesis, colocalization studies were
performed with Mito Tracker Deep Red (MTDR). The spectral properties of MTDR are
complementary to that of LZn(II): LZn(II) is excited at 400 nm and its emission is collected
at 500 to 550 nm, whereas MTDR is excited at 644 nm and emits at 655 nm (Figure 4).
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(b)
(c)
(a)
2 μm
Lyso Tracker Red
2 μm
2 μm
Merged
LZn(II)
Figure 5. Colocalization experiments of intracellular localization of L•Zn(II) using Lyso Tracker probes:
Widefield microscopy images of in cellular emission of L•Zn(II) (panel a) and emission from Lyso Tracker Deep
Red (panel b). The overlap of the intensity is shown in panel c. Panel c shows no overlap of the green and red
fluorescence indicates that L•Zn(II)is not localized over lysosomes. Scale bar 2 μm.
The intensity profile of the widefield images indicates that 60 percent of the MTDR signal
matches that of LZn(II) (Figure 4). Calculated Pearson’s coefficient (93%) also confirmed
that LZn(II) localizes in the mitochondria of RAW cells (Fig. 3 and 3d). For further
confirmation of subcellular localization, similar colocalization experiments were performed
using Lyso Tracker Deep Red (LTDR) and Hoechst 33258. (Figures 5 and 6). These studies
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ensured that LZn(II) does not localize in lysosomes or nuclei. This confirmed that LZn(II)
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(c)
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LZn(II)
Hoechst
Merged
10 μm
10 μm
10 μm
Figure 6. Dual colour SIM using L•Zn(II) in presence of Hoechst33342. Scale bar 10μm. (a) with L•Zn(II); (b)
with Hoechst33342 and (c) is the overlap image. Ext = 400 nm and scale bar 10μm.
After confirming the subcellular localization and specific recognition properties of LZn(II)
in-vitro condition, its ability to detect NPPs in mitochondria of live cells was evaluated. The
most abundant NPP inside the mitochondria is ATP. The reported normal concentration is
∼2-3 mM in healthy mammalian cells, which is at least 5-times higher than those of other
45-46
NPPs, excluding ADP (∼0.7 mM).
Therefore, LZn(II) complex was used to detect
changes in [ATP] within mitochondria during drug-induced apoptosis. Staurosporine (STS)
an indolocarbazole is known to be a potent protein kinase C inhibitor 47-48 that enhances the
ATP concentration in human cells. RAW 264.7 macrophage cell lines were incubated with
STS (5 μM) and then these pre-treated cell lines were further treated with LZn(II). A
distinct growth in luminescence intensity was observed in the mitochondria. Time-lapse
images recorded in the presence of STS revealed a steady increase in intracellular
luminescence intensity over 200 min (Figure 7 a-d). However, insignificant changes in
luminescence intensity were observed in time-lapse images recorded for analogous
experiments performed in the absence of STS (Figure S10). These results confirmed that the
reagent LZn(II) can detect subtle changes in [ATP] or [ATP] + [ADP] in mitochondria for
a period of 210 min during the pre-apoptotic stage (∼3.5hr). This finding is consistent with
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a previously reported result, which indicated that during STS-induced cell death the
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concentration
of
ATP
in
the
cytosol
gradually
increases.
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2.0x103
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1.0x103
5.0x102
0.0
(c)
(d)
Figure 7. Fluorescence analysis of RAW 264.7 macrophage cells stained with L•Zn(II)complex. (a-d)Time-
lapse confocal micrographs of RAW 264.7 macrophage cells stained with 1μM L•Zn(II)complex before (0
min) and after (200 min) treatment with 5 μM STS. Scale bars: 20 μm. (e) Time dependence of the change in
fluorescence intensity upon treatment with 5 μM STS. Data represent the mean ± standard deviation of three
replicates.
4. CONCLUSION
A new Zn(II)-based coordination complex was synthesized. This was found to bind specifically
to NPPs in the presence of all other competing anions and natural amino acids. A significant
enhancement in emission intensity was observed on the binding of NNPs to Zn(II)-centre of
the L•Zn(II). This along with 1H NMR spectroscopic studies were performed in confirming the
binding affinities as well as the binding of the di- or tri-phosphate units of ADP or ATP to the
Zn(II)-centre. This reagent was found to be physiologically benign. Structured illumination
microscopic studies confirmed localization of the reagent in the mitochondrial region of
the RAW 264.7 macrophage cells. This reagent could be utilized for probing the change in
ATP concentration in mitochondria during drug-induced apoptosis. In addition, this
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mitochondria targeting reagent may offer application in the study of intracellular transport
mechanisms and mitochondrial dysfunction based diseases. Using such systems, in-vivo
imaging technologies for live monitoring of various biological process will be possible.
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ASSOCIATED CONTENT
Supporting Information:
The Supporting Information is available free of charge on the ACS Publications website at
DOI: 10.xxxx/acs.cgd.xxxxxxx.
AUTHOR INFORMATION
Corresponding Author
Email: sumitpramanik@csmcri.res.in
James.thomas@sheffield.ac.uk
a.das@csmcri.res.in
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
AD acknowledges SERB grants (EMR/2016/001850 & JCB/2017/000004) and DBT (India)
grant BT/PR22251/NNT/ 28/1274/2017) for financial support. SKP acknowledges SERB
(PDF/2015/000745) for financial support. SS acknowledges MRC Grant for Doctoral
Fellowship.
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A Fluorescent Chemodosimeter for Organelle-Specific Imaging of Nucleoside
Polyphosphate Dynamics in Living Cells
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Harwinder Singh, Sreejesh Sreedharan, Rajeswari Tiwari, Christa Walther, Carl Smythe,
Sumit Kumar Pramanik, Jim. A. Thomas and Amitava Das
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New fluorescence-based reagent for probing subtle changes of NPPs in mitochondria
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