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Letter
Ratiometric imaging of cysteine level changes in endoplasmic reticulum during H2O2-induced redox imbalance Baoli Dong, Yaru Lu, Nan Zhang, Wenhui Song, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01457 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019
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Analytical Chemistry
Ratiometric imaging of cysteine level changes in endoplasmic reticulum during H2O2-induced redox imbalance Baoli Dong, Yaru Lu, Nan Zhang, Wenhui Song and Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, People’s Republic of China * E-mail:
[email protected]. ABSTRACT: Cysteine (Cys) is an important mediator to regulate the redox state of endoplasmic reticulum (ER), and its level is closely related with many ER stress induced serious diseases. Herein, we present an ER-specific fluorescent probe for the ratiometric imaging of cellular Cys, for the first time. The probe exhibited desirable selectivity and sensitivity to Cys. Biological imaging experiments demonstrated that the probe possessed ER-targeting property and showed ratiometric response to Cys in ER, and could be applied for the ratiometric imaging of Cys level changes during H2O2-induced redox imbalance in living cells.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, and serves critical functions in protein folding and secretion, calcium homeostasis and lipid biosynthesis.1-2 Under both normal and pathophysiological conditions, ER stress occurs when suffered from multiple cellular disturbances, and has been well-documented to implicate in many serious diseases including cancer, type 2 diabete, Alzheimer’s disease and Parkinson’s disease.3-6 As a prevalent endogenous reductive thiol, Cysteine (Cys) is recognized as one important mediator to regulate the redox state of ER by means of trapping various reactive oxygen species (ROS) during ER stress.7-8 On the other hand, excessive Cys level is extremely toxic to biological systems mainly due to the abnormally production of homocysteine, acid-base imbalance and oxidative stress.9-11 Excessive Cys could result in the extensive production of vacuoles production in the cytoplasm, promote the upregulation of C/EBP homologous protein (CHOP), and activate ER stress.12 Therefore, in real-time detection of Cys levels in ER environments is critical for the in-depth understanding of its biological functions. Currently, numerous effective methods including electrochemical voltammetry, colorimetric method, spectrophotometry and liquid chromatography have been developed for the detection of Cys.13-15 These methods are usually not suited for the in situ detection of Cys in living systems. By contrast, fluorescence imaging is a very attractive technique for detecting biomolecules in living systems because of excellent sensitivity, in situ detection manner and real-time analysis.16-18 To date, many fluorescent probes for the detection of Cys in biological systems have been developed.1923 Thereinto, most of these probes have no ER targeting group and are not suitable for the selective imaging of Cys in ER environments, and currently the ER-specific probe for Cys is
very scarce. Meng et al developed a turn-on probe for monitoring Cys levels in ER, which based on the decomplexation of Cu2+ ions by thiol and displayed only one fluorescence signal in response to Cys.7 Compared with turnon type probe, ratiometric probes utilize the ratio value of the intensities at two different wavelengths as the signal output, and are less prone to be interfered with the disturbances including the variations of probe concentration, excitation intensity, etc.24 However, to the best of our knowledge, the ER-specific ratiometric fluorescent probe for the imaging of cellular cysteine has not been developed. Therefore, it is important to fabricate ER-specific ratiometric probes for the imaging of Cys in living systems. Herein, we present a new ER-specific fluorescent probe for the ratiometric imaging of cellular Cys, for the first time. Naphthalimide fluorophore was initially selected as the signal reporter because of its numerous advantages including excellent photostability and high fluorescence quantum yield. p-Toluenesulfonamide was employed as ER-specific group, and embedded into a naphthalimide fluorophore to endow it with ER-targeting property.25-27 Due to the high reactivity to Cys over the other relevant biological
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Scheme 1. Structure of ER-targeting ratiometric fluorescent probe (NapCys) and its response mechanism to Cys.
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Analytical Chemistry thiols, acrylate was used as the Cys-responsive site.28-29 With these considerations in mind, we fabricated an ER-specific ratiometric probe (Nap-Cys, Scheme 1) for the ratiometric imaging of Cys in ER of the living cells. The synthesized probe Nap-Cys was characterized by 1H NMR, 13C NMR, HRMS and LCMS (Supporting information). With the probe Nap-Cys in hand, we initially assessed its optical properties by UV-Vis absorption and fluorescence spectra. As shown in Figure S1, Nap-Cys exhibited a main absorption band peaked at 350 nm with molar extinction coefficient (ε) of 24000 cm-1.M-1, which can be ascribed to the π-π* transition of Nap-Cys. After the treatment of Nap-Cys with 50 or 100 μM Cys, a new absorption peak at 450 nm appeared and the solution color changed from colorless to light yellow, indicating that the reaction between the probe and Cys indeed occurs. Under excitation at 390 nm, Nap-Cys displayed an emission band at 440 nm in PBS (pH = 7.4, 10% EtOH, 20 mM) (Figure 1A). However, upon addition of Cys with increasing concentrations, the emission at 440 nm descreased tardily, while a new emission band at 550 nm appeared and increased gradually, and the ratio value of the emission intensities at 550 nm and 440 nm (I550/I440) arised from 0.085 to 1.86, representing an approximate 22-fold ratiometric signal response (Figures 1A and 1B). The ratio value I550/I440 exhibited excellent linear relation with Cys concentration in the range 10-80 μM (R2 = 0.9914), and the detection limit was calculated to be 1.80 μM (Figure S2). Time-dependent fluorescence spectra suggested that the emission of Nap-Cys showed nearly no change under excitation at 390 nm for 40 min, demonstrating the photostability of the probe (Figure 1C and Figure S3). After (B) 2.0
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Figure 1 (A) Fluorescence spectra of 5 μM Nap-Cys upon addition of 0200 μM Cys in PBS (pH = 7.4, 10% EtOH, 20 mM). (B) Fluorescence intensity ratios (I550/I440) of Nap-Cys treated with various Cys concentrations. (C) Fluorescence spectra of 5 μM Nap-Cys treated with (red) and without (black) 200 μM Cys at different times. Inset: Fluorescent images of the probe Nap-Cys before and after treated with Cys for 20 min under UV irradiation. (D) Fluorescence spectra of 5 μM Nap-Cys upon addition of various relevant analytes. Concentration: GSH, 10 mM; other analytes, 100 μM. λex = 390 nm.
the addition of Cys to Nap-Cys, time-dependent fluorescence spectra suggested that Nap-Cys could show fast response to Cys within 20 min (Figure 1C and Figure S4). Meanwhile, it
can be clearly observed that the fluorescence color of NapCys changed from blue to yellow after the addition of Cys for 20 min. Therefore, Nap-Cys could be potentially used for the ratiometric imaging of Cys in biological systems. Next, we verified the response mechanism of Nap-Cys to Cys by optical spectra of HRMS assay. The compound NapOH displayed a main absorption peak located at 440 nm, which was identical with the emerged absorption after the response of Nap-Cys to Cys (Figure S5). Under excitation at 390 nm, Nap-OH showed an emission band at 550 nm, consisting with the clearly increased emission band after the treatment of Nap-Cys with Cys. Meanwhile, HRMS data showed that the peaks at 411.1012 and 433.0829 corresponding to Nap-OH (cald. [M+H]+ 411.1009 and [M+Na]+ 433.0829) could be observed when Nap-Cys responded to Cys (Figure S6). Therefore, it can be concluded that the compound Nap-OH is the product after Nap-Cys reacts with Cys, and the response mechanism of Nap-Cys to Cys bases on the cleavage of ester to hydroxyl group. Given that the electron-donating capacity of hydroxyl (-OH) is stronger than that of ester (-OCOR), the converion of ester to hydroxyl endows the naphthalimide fluorophore with a typical “push-pull” structure. Namely, the red shift of the absorption and emission of Nap-OH relative to that of Nap-Cys could be ascribed to intramolecular charge transfer (ICT) effect. The selectivity of Nap-Cys to Cys was evaluated by the fluorescence spectra of Nap-Cys in the presence of a wide variety of biologically relevant species including GSH, Hcy, H2S, glucose and vitamin C (VC). Under excitation at 390 nm, only Cys could trigger marked emission ratio (I550/I440) changes, indicating that Nap-Cys possesses high selectivity for Cys over the other competing biological species (Figure 1D and Figure S7). Under pH 4.0-9.0, the fluorescence spectra of Nap-Cys showed nearly no changes, indicating that this probe was stable at weak or acid environments (Figure S8 in ESI†). After the treatment with Cys, the emission ration (I550/I440) showed obvious increase at pH 6.0-9.0, and it suggested that Nap-Cys could show desirable response to Cys at physiological conditions (pH 7.4) and ER condition (pH 7.2). Taken together, Nap-Cys could act as a ratiometric probe for the detection Cys in living systems. Subsequently, we explored the feasibility of Nap-Cys to image Cys in ER of living cells. MTT data demonstrated that Nap-Cys had low cytotoxicity to living HeLa cells below the concentration of 20 μM (Figure S9). The distribution of NapCys at subcellular levels was then checked by colocalization experiments with commercial ER, mitochondria and lysosome organelle trackers using HeLa cells. After pretreated with 5 μM Nap-Cys for 30 min, the HeLa cells were stained with 1 μM ER-Tracker Red, 1 μM LysoTracker Red and 1 μM MitoTraker Red for 10 min, respectively. As shown in Figure 2, the green fluorescence from Nap-Cys exhibited excellent overlap with the red fluorescence from ER tracker, and the Pearson’s colocalization coeffcient (R) value of Nap-Cys with ER-tracker is 0.94. By contrast, the overlaps between the
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Figure 2 Images of HeLa cells costained with 5 μM Nap-Cys for 30 min, and then treated with 1 μM ER-tracker red, 1 μM MitoTracker red and 1 μM LysoTracker Red for 10 min, respectively. Blue channel (425-475 nm), λex = 405 nm; Green channel (570-620 nm), λex = 561 nm. Scale bar = 5 µm.
green fluorescence from Nap-Cys and the red fluorescence from LysoTracker Red or MitoTracker Red were relatively weak. The R values of Nap-Cys with LysoTracker Red and MitoTracker Red are 0.29 and 0.57, respectively. Therefore, Nap-Cys was distributed in cellular ER and could serve as an ER-targeting probe. The feasibility of Nap-Cys to image endogenous Cys in living cells was then explored. After the incubation of HeLa cells with 5 μM Nap-Cys, weak blue fluorescence and strong green fluorescence could be observed clearly (Figure 3A). Nethylmaleimide (NEM), a well-known thiol-blocking agent, is commonly used for the consumption of cellular thiols.30 When
and subsequently incubated with Nap-Cys for 20 min, it can be found that the blue fluorescence was diminished while the green fluorescence increased obviously (Figures 3C and 3E). These results of fluorescence images were further verified using flow cytometry analysis (Figures 3D and 3F). Such findings demonstrated that Nap-Cys could be applied for the ratiometric imaging Cys in the ER of living cells. To the best of our knowledge, the ER-specific ratiometric probe for the imaging of cellular Cys, described herein, was reported for the first time. Redox imbalance is usually caused by the excess generation of ROS or the decrease in protective antioxidants, and has close relation with inflammation, cancer and 31-32 neurodegenerative diseases. As an important endogenous antioxidant, Cys plays critical roles in maintaining cellular redox balance.33-34 By altering the redox balance with H2O2, we investigated the fluorescence response of Nap-Cys to cellular cysteine level changes. The HeLa cells only treated with 5 μM Nap-Cys displayed weak blue fluorescence and strong green fluorescence because of the response of Nap-Cys to endogenous Cys (Figure 4A). When HeLa cells were pretreated with 200 μM or 400 μM H2O2, and then incubated with 5 μM Nap-Cys for 20 min, it can be observed that the blue fluorescence increased and the green fluorescence was diminished slightly (Figures 4B, 4C and 4E). Flow cytometry analysis also determined the changes of fluorescence in blue and green channels (Figures 4D and 4F). It indicates that the addition of
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Analytical Chemistry
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Figure 3 (A) HeLa cells treated with 5 μM Nap-Cys for 20 min. (B) HeLa cells pretreated with 1 mM NEM for 30 min and then treated with 5 μM Nap-Cys for 20 min. (C) HeLa cells pretreated with 1 mM NEM for 30 min and then treated with 100 μM Cys for 30 min, and subsequently incubated with 5 μM Nap-Cys for 20 min. Blue channel (425-475 nm), Green channel (570-620 nm), λex = 405 nm. Scale bar = 20 µm. (D) Flow cytometry analysis of the cells in panels (A, B and C). (E) The average ratio values in panels A-C. (F) The average ratio values in panel D.
HeLa cells was pretreated with 1 mM NEM and subsequently incubated with Nap-Cys for 20 min, the blue fluorescence increased while the green fluorescence was weakened (Figure 3B). In addition, when HeLa cells were pretreated with 1 mM NEM for 30 min and then treated with 100 μM Cys for 30 min,
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Figure 4 (A) HeLa cells treated with 5 μM Nap-Cys for 20 min. (B) HeLa cells pretreated with 200 μM H2O2 for 20 min and then treated with 5 μM Nap-Cys for 20 min. (C) HeLa cells pretreated with 400 μM H2O2 for 20 min and then treated with 5 μM Nap-Cys for 20 min. Blue channel (425475 nm), Green channel (570-620 nm), λex = 405 nm. Scale bar = 25 µm. (D) Flow cytometry analysis of the cells in panels (A, B and C). (E) The average ratio values in panels A-C. (F) The average ratio values in panel D.
H2O2 to cells could cause the decrease of intracellular Cys level probably due to the oxidation of H2O2 to Cys. In addition, the probe Nap-Cys also could show fluorescence response to cellular Cys within 20 min (Figure S10). Therefore, Nap-Cys could serve as an effective tool for the detection of Cys level changes in living cells.
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In conclusion, we have developed a new ER-specific ratiometric fluorescent probe (Nap-Cys) for the imaging of cellular Cys. The probe Nap-Cys employed ptoluenesulfonamide as ER-specific group, and utilized acrylate as the Cys-responsive site. After responding to Cys, Nap-Cys afforded remarkable fluorescence ratiometric signal changes. Meanwhile, the probe showed desirable selectivity to Cys over the other common thiols. Biological imaging experiments demonstrated that Nap-Cys possessed ER-targeting property and displayed ratiometric response to Cys in ER, and could be applied for the ratiometric imaging of cellular cysteine level changes during H2O2-induced redox imbalance. We expect that the ER-specific ratiometric Cys probe could be further applied to the in-depth study of Cys-associated physiological and pathological progresses.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details, synthesis of the probe, absorption and fluorescence spectra, characterization data.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected].
Notes The authors declare no conflict of interest.
ACKNOWLEDGMENT This work was financially supported by NSFC (21472067, 21672083, 21877048, 51602127), Taishan Scholar Foundation (TS 201511041), and the startup fund of the University of Jinan (309-10004).
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