Biosensors and Bioelectronics 68 (2015) 189–196
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Imaging of nucleolar RNA in living cells using a highly photostable deep-red fluorescent probe Bingjiang Zhou 1, Weimin Liu 1, Hongyan Zhang, Jiasheng Wu, Sha Liu, Haitao Xu, Pengfei Wang n Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 November 2014 Received in revised form 23 December 2014 Accepted 24 December 2014 Available online 26 December 2014
A new crescent-shape fluorescent probe (named here as CP) that selectively stains RNA in nucleoli of living cells is prepared. CP shows a deep-red emission (658 nm) and a large Stokes shift because of the introduction of rigid-conjugated coumarin moiety into the molecular structure. Cell imaging experiments indicate that CP can rapidly stain nucleoli in living cells by binding with nucleolar RNA, showing performance superior to commercially available nucleoli dye SYTO RNASelect in terms of high photostability and selectivity. More significantly, these excellent properties together with low cytotoxicity enable CP to monitor nucleolar RNA changes during mitosis, and after treating with anti-cancer drugs cisplatin, actinomycin D and α-amanitin. Thus, CP could be a potential tool for real-time, long-term visualization of the dynamic changes for nucleolar RNA and evaluation of the therapeutic effect for anticancer drugs that targeted RNA polymerase I (Pol I). & 2014 Elsevier B.V. All rights reserved.
Keywords: RNA Nucleoli Fluorescent imaging Real-time
1. Introduction RNA molecules are responsible for a wide range of functions in living cells, from physical conveyance and interpretation of genetic information, structural support for molecular machines, and regulation/silencing of gene expression, to essential catalytic roles (Prasanth and Spector, 2007; Storz et al., 2005). The ability to acquire complete spatial–temporal profiles of RNA synthesis, processing, and transport is therefore critical to understand cell function and behavior in health and disease conditions (Bao et al., 2009). In addition, most of the RNA in the nucleus is localized to the nucleolus, where is the key site of ribosomal RNA (rRNA) transcription, processing, and ribosome assembly (Yusupov et al., 2001). As one of the most powerful techniques for monitoring bimolecular in living systems, fluorescence imaging techniques are widely used to visualize morphological details of RNAs in cells. Those include microinjection of fluorescently tagged RNA molecules (Mhlanga et al., 2005; Wilkie and Davis, 2001), fluorescence in situ hybridization (Guo et al., 2012; Simon et al., 2010; Volpi and Bridger, 2008), molecular beacons (Chen and Tsourkas, 2009), and small-molecule fluorescent probes (Li and Chang, 2006; Liu et al., n
Corresponding author. E-mail address: [email protected] (P. Wang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.12.055 0956-5663/& 2014 Elsevier B.V. All rights reserved.
2011; O’Connor et al., 2009). Among these, small-molecule fluorescent probes are the most practical tool for live cell imaging, because the good cellular permeability make it does not require fixation of cells which can disturb the cell membrane and organelles. Considerable efforts have been exerted to develop small-molecule fluorescent probes for RNA imaging in living cells. However, in comparison with DNA probes, RNA probes, especially nucleolar RNA probes for live cell imaging are quite rare due to better affinity for DNA and poor nuclear membrane permeability. In 2006, Chang et al. first reported three selective RNA-binding styryl probes screened from 88 compounds through RNA assay and live cell imaging methods (Li and Chang, 2006). Recently, Song and Liu et al. also find other small-molecule probes for RNA in nucleoli and cytoplasm based on styryl dyes (Liu et al., 2014; Song et al., 2014). In addition, Stevens et al. designed an energy transfer probe for duplex RNA in cellular imaging (Stevens et al., 2008). Peng et al. reported a near-infrared RNA fluorescent probe for imaging living cells assisted by the macrocyclic molecule CB7 (Li et al., 2013). So far, the only commercially available nucleolar RNA probe is a green fluorescent dye known as “SYTO RNASelect”, whose molecular structure has not been published to date. However, these reports on nucleolar RNA staining also have problems of poor photostability, high cytotoxicity, short emission wavelength (o 600 nm), or poor cell permeability. Thus it is highly desirable to develop
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new fluorescent probes with excellent performance to monitor the changes of nucleolar RNA. The nucleolus is a subnuclear organelle that contains ribosomal RNA gene clusters and ribosome biogenesis factors. In this study, a fluorescent probe CP was development by rigid hybridizing coumarin and pyronin moieties to form crescent-shape structure. The intramolecular charge transfer reservation and planarity amplification resulted in large Stokes shift and deep red emission. Then CP was found to be a potential fluorescent probes for imaging RNA distribution within the nucleolar. The photophysics properties of CP and its interaction with nucleic acid (NA) have been investigated through absorption spectroscopy and fluorescence spectroscopy. Moreover, we successfully used CP to study the details of nucleolar RNA dynamic changes during mitosis, as well as after treatment with inhibitor of RNA polymerase I (Pol I).
2. Experimental section 2.1. Materials and apparatus All commercial chemicals were used without further purification. 7-amino-1-methyl-1,2,3,4-tetrahydroquinoline hydrochloride was purchased from HangZhou Trylead Chemical Technology Co., Ltd. Escherichia coli total RNA and Calf thymus (CT) DNA were obtained from Sigma-Aldrich. The stock solutions of CP were prepared in DMSO (1.0 10 2 M). All UV–vis and fluorescence spectra in this work were recorded using Hitachi U3900 and Hitachi F4600 fluorescence spectrometers, respectively. All measurements were carried out at room temperature. Water was purified by a Millipore filtration system. 1H NMR (400 MHz) and 13 C NMR (101 MHz) spectra were collected using a Bruker Advance 400 spectrometer with tetramethylsilane as an internal standard. Electrospray ionization high resolution mass spectra (ESI-HRMS) were obtained usi a Bruker Apex IV FTMS. Fluorescent images were acquired with a Nikon C1si laser scanning confocal microscope, equipped with a live-cell incubation chamber maintaining a humidified atmosphere of 37 °C and 5% CO2. Images were processed by NIS-element analysis AR 4.13.00. Clear nucleoli of Hela cells amplification imaging were obtained by a Delta Vision OMX imaging system.
2.2. Synthesis CP can be readily prepared by condensation reaction of 3-aldehyde-4-chlorocoumarin derivates and 3-aminophenol. Detailed synthetic routes and methods are shown in Scheme 1 and Supplementary information. CP: 1H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H), 8.01 (d, J¼ 9.5 Hz, 1H), 7.74 (s, 1H), 7.35 (d, J ¼9.3 Hz, 1H), 7.23 (s, 1H), 6.68 (s, 1H), 3.55 (s, 2H), 3.36 (s, 6H), 3.16 (s, 3H), 2.81 (s, 2H), 1.93 (d, J ¼10.8 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 161.61, 158.36, 158.14, 157.00, 156.54, 154.15, 145.09, 133.80, 122.81, 122.63, 116.27, 115.74, 105.39, 99.56, 97.34, 96.46, 50.97, 40.96, 26.34, 20.44. ESI-HRMS m/z calcd for [C22H21N2O3] þ 361.15467, found 361.15508. 2.3. Determination of quantum yield Fluorescence quantum yields (ΦU) are measured using cresyl violet in methanol as the reference (ΦR ¼ 0.54) based on the following equation:
⎛ A ⎞⎛ n 2 ⎞ ΦU = ΦR⎜ U ⎟⎜⎜ U2 ⎟⎟ ⎝ AR ⎠⎝ nR ⎠ where AU and AR are the integrated areas under the corrected fluorescence spectra for the sample and reference, nU and nR are the refractive indexes of the sample and reference, respectively (Magde et al., 1979). 2.4. Cell culture and staining Hela, A549 and L929 were gifted from the center of cells, Peking Union Medical College. L929, A549 or Hela cells were grown in RPM1640, McCoy's 5A or Dulbecco's modified Eagle's medium (DMEM) respectively, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated under 5% CO2 at 37 °C. Cells were seeded on confocal dish for imaging 12–24 h prior to conduction of experiment. Below is a general procedure for labeling living cells. Cells were adhering to coverslips. CP was dissolved in DMSO to a final concentration. After added the appropriate of dye stock solution directly to the culture medium, cells were incubated for 20 min at 37 °C in 5% CO2, and then imaged in dye medium at 37 °C in 5% CO2 without wash. For colocalization experiments, CP was
Scheme 1. Synthesis of CP. Conditions: (a) 85% phosphoric acid, reflux, 24 h; (b) diphenyl malonate, toluene, reflux, 6 h; (c) phosphoroxychloride, 50 °C, 30 min; and (d) 3aminophenol, acetic acid, 85 °C, 2 h, perchloric acid .
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incubated with Hela cells in DMEM for 20 min, and then the medium were replaced with fresh medium in the presence of SYTO RNASelect for 20 min. The cover slips were washed by PBS twice and imaged.
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of fresh medium containing 5 mM CP. After incubated at 37 °C, 5% CO2 for 30 min, the dish was placed on the microscope platform with a live-cell incubation chamber. Fluorescence images at Cy5 channel were acquired every 4 min to capture the dynamic process of nucleoli in cells undergoing cell division.
2.5. DNase and RNase treatment 2.9. Drug treatment Hela cells were fixed in pre-chilled methanol at 20 °C for 1 min. The cell membrane was permeablized with 1% Triton X-100 in PBS for 1 min at room temperature. After rinsing with PBS twice, 0.5 μL 10 mM CP stock solution and 1 mL PBS solution were added. Cells were then incubated in this dye PBS solution for 20 min at room temperature before being rinsed by clean PBS twice. A total of 1 mL clean PBS (as control experiment), 20 u/mL DNase (Takara), or 25 mg/mL DNase-Free RNase (Takara) were added into the three other dishes and incubated at 37 °C in 5% CO2 for 2 h. Cells were rinsed again by clean PBS twice before imaging. The fluorescent imaging pictures were obtained by using an equal exposure time for control, DNase, and RNase experiments.
Hela cells were seeded in 35 mm dishes and grown until they were 75–80% confluent. Culture medium was replaced by 2 mL of fresh medium containing 5 mM CP. After incubated at 37 °C, 5% CO2 for 30 min, the cells were treated with anticancer drugs (cisplatin (40 mM), actinomycin D (0.05 mg/mL) or α-amanitin (10 mg/mL)). The dish was placed on the microscope platform with a live-cell incubation chamber during imaging processes. Fluorescence images at Cy5 channel were acquired every 20 min, lasting 7 h (cisplatin), 6 h (actinomycin D), 3 h (α-amanitin), respectively.
3. Results and discussion 2.6. Cytotoxicity test 3.1. Synthesis and characterization of CP CP stock solutions were diluted by fresh medium into three desired concentration (1, 3, 5, 7, 10 μM). Hela cells were cultured in a 96-well plate for 12–24 h before the conduction of the experiments. The cell medium was then exchanged by different concentrations of CP medium solutions. They were then incubated at 37 °C in 5% CO2 for 24 h before cell viability was measured by the MTT assay. The cell medium solutions were exchanged by 100 μL of fresh medium, followed by the addition of 20 μL (5 mg/mL) MTT solution to each well. The cell plates were then incubated at 37 °C in 5% CO2 for 4 h. Absorbance was measured at 570 nm. The absorbance measured for an untreated cell population under the same experimental conditions was used as the reference point to establish 100% cell viability. Duplicated experiments have been tested. 2.7. Photostability test Hela cells were incubated in 1 mL culture solution consist of 5 μM CP or 1 μM RNASelect for 20 min and imaged by confocal microscope (Nikon C1si) without wash. The laser powers 488 nm and 640 nm were used to irradiate the RNASelect and CP stained cells respectively and the fluorescence were collected at FITC channel for RNASelect and Cy5 for CP by no delay scan mode. The initial intensity referred to the first scan of each dye. 2.8. Nucleolar dynamic observations during mitosis Hela cells were seeded in 35 mm dishes and grown until they were 75% to 80% confluent. Culture medium was replaced by 2 mL
The crescent-shape probe CP can be readily prepared by condensation reaction of 3-aldehyde-4-chlorocoumarin derivates and 3-aminophenol. Detailed synthetic routes and methods are shown in Scheme 1. Compound 2 was acquired by interesterification and Friedel–Crafts reaction of compound 1 with diphenyl malonate, whereas compound 3 was synthesized by Vilsmeier–Haack reaction. Eventually, CP was obtained from the condensation reaction of compound 3 with 3-aminophenol. The overall chemical structure of CP was confirmed via 1H NMR, 13C NMR, and ESI-HRMS (Fig. S1). 3.2. Photophysics properties Absorption and fluorescence spectra of CP in CH2Cl2 and Tris– HCl–EDTA (TE) buffer solution (pH ¼7.4) were investigated (Fig. 1). Corresponding photophysical data are summarized in Table S1. The maximum absorption of CP is 598 nm, and the maximum emission is 658 nm in TE buffer solution. Larger Stokes shift (60 nm) of CP in TE buffer solution is observed compared with that of xanthene in aqueous solution, which could reduce self-absorption, as well as obtain high-resolution and low-detection limits. Fluorescence quantum yield of CP is 0.22 in CH2Cl2 and only 0.01 in TE buffer solution (Table S1). The striking contrast of fluorescence quantum yields in different polar solvents indicating CP is sensitive to environments. In addition, the pH effect (5–8) of CP was also investigated (Fig. S2), and sensitivity to acid conditions was observed for CP. This may attribute to the containing of a
Fig. 1. Photophysical properties of CP. (A) Absorption and (B) fluorescence spectra of CP (10 μM) in CH2Cl2 and Tris–HCl–EDTA (TE) buffer solution (pH ¼7.4).
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Fig. 2. Fluorescent images of living cells by CP. (A) CP (5 μM) was incubated with Hela cells in Dulbecco's modified Eagle's medium (DMEM) for 20 min and imaged. (B) Fluorescence intensity profile of selected region in (A). (C) Clear nucleoli amplification imaging of Hela cells with 5 μM CP by Delta Vision OMX system. (D) Fluorescence intensity profile of selected region in (C). (E) A549 cells incubated with 5 μM CP for 20 min in McCoy's 5A and imaged. (F) L929 cells incubated with 5 μM CP for 20 min in RPM1640 and imaged. Images excited at 640 nm laser power, Cy5 channel.
positive charge in molecular structure (Arden–Jacob et al., 2001; Wang et al., 2010). 3.3. Fluorescent imaging properties in living cells To examine the cellular permeability/uptake, laser confocal imaging studies of CP were carried out in different cells, including tumor cells (HeLa and A549 cells) and normal cells (L929 cells) (Fig. 2). A red semiconductor laser (640 nm) was used as the light source for cell imaging and this could avoid cellular autofluorescence interference. After incubation with CP (5 μM) for 20 min, a clear nucleolar staining in the nucleus and some bright spots in the cytoplasm were observed for CP (Fig. 2A). No obvious cell morphology or viability change was found during the imaging. The fluorescence intensity in the nucleoli is about 30 times higher than that in the nucleus (Fig. 2B), and high signal/noise ratio achieved a strong contrast for simultaneous visualization of the nucleus and nucleolus. A clear nucleolar amplification imaging was also obtained by the Delta Vision OMX imaging system. Nucleolar fluorescence exhibited a honeycomb structure with different apertures and micro-holes (Fig. 2C). Variational intensity in region holes also indicated uneven distribution of CP in the nucleoli (Fig. 2D). Similar staining is observed in A549 and L929 cells (Fig. 2E and F), indicating that CP has good nuclear membrane permeability in cancer and normal cells. 3.4. Cell localization and the interaction with NA A commercially available nucleolar stain SYTO RNASelect was used to co-stain cells with CP to confirm that the observed bright
spots in the nucleus were related to the fluorescence of CP that localized at the nucleoli. As we know, the nucleolus contains abundant proteins and RNAs, and SYTO RNASelect stained RNA in nucleoli. Orange fluorescence indicated the same subcellular localization of the nucleoli in SYTO RNASelect and CP (Fig. 3A). The fluorescence throughout the cytoplasm was predominantly associated with lysosome by co-staining with LysoTracker Green (Fig. S3). The brighter fluorescence of CP in lysosome may result from acidic environment with the lysosomal pH. This might be attributed to lysosomes are acidic organelles. In addition, deoxyribonuclease (DNase) and ribonuclease (RNase) digest experiments were also performed to identify the stained species by CP in the nucleoli. DAPI and SYTO RNASelect were tested as controls. Upon treatment with DNase (Fig. 3B), no significant loss of fluorescence in the nucleoli occurred for CP. By contrast, after RNase digestion, the nucleolar fluorescence signals of CP were completely lost and redistributed in the nucleus. CP exhibited a similar behavior in the digest experiment with SYTO RNASelect, indicating that nucleolar fluorescence originated from the binding of CP with RNA in the nucleoli. To further investigate the CP's preference to RNA in vitro, absorption and fluorescence spectra of CP upon nucleic acid (NA) binding were investigated in buffer solution (Fig. 4 and Table S2). As Fig. 4A shows, upon addition of NA at a 100:1 CNA/CCP ratio, a pronounced bathochromic effect in absorption spectra (above 20 nm) is observed for CP, which reveal the strong interaction between CP and NA. The fluorescence intensity of CP upon RNA displays a larger fluorescence enhancement than DNA for the same amount of NA (Fig. 4B). The result might be attributable to crescent-shape ligand with positive charges may prefer binding with
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Fig. 3. Cell Localization of CP. (A) Co-localization of SYTO RNASelect (1 μM). (B) DNase and RNase digest experiments. Images of CP (0.5 μM), as well as DAPI (1 mg/ml) and SYTO RNASelect (0.5 mM), are shown as comparison experiments. Equal exposure was used for the same dye imaging. DAPI (blue: DAPI channel), SYTO RNASelect (green: FITC channel), and CP (red: Cy5 channel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Spectra changes of CP in the present of NA. (A) Absorption and (B) fluorescence spectra of CP (10 μM) in TE buffer (pH¼ 7.4) at 100:1 CNA/CCP ratio.
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RNA (Cetinkol and Hud, 2009; Sinha and Kumar, 2009). A similar phenomenon for SYTO RNA-Select was also observed in buffer solution (Fig. S4), indicating that CP and SYTO RNA-Select both have higher response to RNA than to DNA in solution. However, CP showed a better selectivity for RNA in nucleus in living cell experiments, which may be possibly due to easy accumulation in nucleolus for CP and the difference between structure of NA in solution and their real state in cells (Li and Chang, 2006; Song et al., 2014). Additionally, the selectivity of CP for RNA was evaluated by the fluorescence intensity response in TE buffer solution. As illustrated in Fig. S5, the other biological molecules including Lcysteine, histidine, glucose, adenosine triphosphate (ATP), Mg2 þ , and Cl do not give rise to significant increase in fluorescence intensity at 658 nm. NA is important biological macromolecules that function in encoding, transmitting, and expressing genetic information. The simultaneous visualization of DNA and RNA in nucleus is important using different dyes during the cell imaging. We investigated the counterstaining behavior for CP with DNA dye Hoechst33342 (Fig. 5). The blue nuclear stain in the DAPI channel and the red nucleolar stain in the Cy5 channel can be clearly visualized. The result also indicates that CP has excellent counterstaining compatibility with Hoechst33342. 3.5. Cytotoxicity and photostability Cytotoxicity and photostability are two important factors for evaluating applicability of new fluorescent probes in live cell imaging. The cytotoxicity of CP toward HeLa cell lines was studied using standard MTT assay after 24 h of incubation (Fig. 6A). CP
exhibited good cell tolerability at imaging concentration after 24 h of incubation. The photostability of CP and SYTO RNASelect in cell imaging were also examined. CP exhibited better photostability than SYTO RNASelect. The fluorescence signals of SYTO RNA-Select decreased by 80% after 52 scans (Fig. 6B). By contrast, no significant decrease in intensity was observed under continuous irradiation for 300 scans in 10 min, indicating that CP is a promising dye for long-term nucleolar RNA imaging. 3.6. Dynamic changes of nucleolar RNA during mitosis Cells have to double in size before dividing, and cell growth correlates with rRNA synthesis. In the cell cycle, the nucleolus is assembled at the end of mitosis and throughout interphase, and disassembles in prophase. Transcription is silenced during mitosis, gradually increases during G1-phase, and reaches maximal levels at S- and G2-phase (Russell and Zomerdijk, 2006). Nucleolar assembly of the rRNA processing study in living cells usually utilize green fluorescence protein-tagged nucleolar proteins and linear oligonucleotide probe (Molenaar et al., 2001; Savino et al., 2001). However, no small organic probe was used in monitoring the dynamic process of the RNAs in the nucleolus during mitosis. With good cell permeability, good photostability, and low cytotoxicity, CP was successfully used in monitoring the temporal and spatial changes of the nucleolar RNA in Hela cells during mitosis (Movie S1). Cells were stained with CP and imaged with timelapse. Fig. 7 shows a 7 h laser scaning confocal experiment that was recorded through a 60x objective in a cell preceded through mitosis. Fluorescence is clearly observed in two large size nucleoli at interphase (Fig. 7A–C), indicating high transcriptional activity in the
Fig. 5. Live cell counterstain CP (5 μM) with Hoechst33342 (1 μM). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 6. Cytotoxicity and photostability of CP. (a) Cytotoxicity of CP; (b) photostability of CP (5 μM) and SYTO RNASelect (1 μM) with increasing number of scans during cell imaging.
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Fig. 7. Dynamics of nucleoli of stained HeLa cells by CP during mitosis. (A–I) Time-dependent fluorescence imaging after addition of 5 mM CP.
Fig. 8. Time-lapse imaging of nucleolus RNA in HeLa cells with cisplatin, Act D, or α-amantin treated using CP (5 μM) in the range of hours.
nucleoli. After about three hours, the cell entered mitosis, rDNA transcription was repressed, and RNA synthesis ceased (Fig. 7C). In the next one hour, the nucleoli were distorted, reducing in size and disappearing, and no longer observed throughout mitosis, along with decreasing fluorescence in the nucleolar region and finally
fading away (Fig. 7D–G). During telophase, the nucleoli rapidly assemble due to the restoration of rDNA transcription and functional activity. As shown in Fig. 7, very small nucleoli were observed and increased in size along with daughter cells, concomitantly with increasing fluorescence intensity in the nucleoli.
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The mitosis time of HeLa cells is about 1.5 h. Then cell enter early interphase, which three or four nucleoli visualized in each nucleus (Yamauchi et al., 2007). A similar observation can also be obtained in normal L929 cells (Movie S2). The results further confirm that CP does not disturb cell proliferation and is expecting to be used in localization of nucleolar RNA dynamic process. Supplementary material related to this article can be found online at. http://dx.doi.org/10.1016/j.bios.2014.12.055 3.7. Dynamic changes of nucleolar RNA under treatment with anticancer drug As a potential tool for real-time, long-term monitoring of the dynamics of RNA in the nucleolus, an important application of CP is to study the mechanism of drug action and evaluate therapeutic effect. A new therapeutic approach to kill cancer cells is based on the specific inhibition of RNA polymerase I (Pol I) activity (Montanaro et al., 2013). Pol I is located in the nucleolus, and its sole function is the transcription of genes encoding rRNA, accounting for at least 60% of cell transcriptional activity. Almost all signaling pathways that affect cell growth and proliferation directly regulate rRNA synthesis, and their downstream effectors converge at the Pol I transcription machinery. The activity of Pol I is related to the synthesis rate and the amount of RNAs in the nucleolus. Real-time monitoring of RNA changes in the nucleolus during cell apoptosis caused by inhibited Pol I will provide valuable insights to drug mechanism and detection of therapeutic efficiency. Cisplatin (cis-diamminedichloroplatinum (II)) is the flagship compound for the treatment of a wide range of cancers. Targets for DNA and redistribution of RNA Pol I have important functions in the clinical success of cisplatin (Jordan and Carmo-Fonseca, 1998). We used CP to monitor nucleolar RNA changes induced by cisplatin. As Fig. 8 shows, no obvious changes in control experiments occurred (Movie S3). However, nucleolus was packaged into granules after 5 h in cisplatin-treated HeLa cells because of the redistribution of RNA Pol I (Fig. 8 and Movie S4). This result is well consistent with the silver staining method (Horky et al., 2001). Similarly, the decreased amount of nucleolar RNA resulted in weaker fluorescence in the nucleolus in the presence of actinomycin D (Act D) due to inhibition of RNA Pol I (Fig. 8 and Movie S5) (Hofgartner et al., 1979). Conversely, the use of α-amanitin, an inhibitor of RNA polymerase II and III which localize in nucleus and cytosol, resulted in no distinct changes of RNA amount in the nucleolus (Fig. 8 and Movie S6) (Rudd and Luse, 1996). The results further demonstrate that CP does target nucleolus RNA and can be used as an imaging tool to evaluate the therapeutic effect of Pol I targeted anticancer drugs.
4. Conclusion In summary, a crescent-shaped probe CP that contains coumarin and pyronin Y-fused conjugated skeleton was designed and synthesized. With good cell permeability, low cytotoxicity, and high photostability, CP can be successfully used in real-time monitoring of nucleolar RNA dynamics during mitosis and apoptosis induced by anticancer drugs that target RNA Pol I. These results provide useful guidelines for CP as a powerful tool for studying the dynamic changes of nucleolar RNA in response to
external stimuli, including anti-cancer drugs, stress, temperature, and so on.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 21373250, 61227008, and F040801). We would like to thank the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science for our Structured Illumination Microscopy work and we would be grateful to Shuoguo Li for her help of analyzing EM images.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.055.
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Imaging of nucleolar RNA in living cells using a highly photostable deep-red fluorescent probe Bingjiang Zhou 1, Weimin Liu 1, Hongyan Zhang, Jiasheng Wu, Sha Liu, Haitao Xu, Pengfei Wang n Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 November 2014 Received in revised form 23 December 2014 Accepted 24 December 2014 Available online 26 December 2014
A new crescent-shape fluorescent probe (named here as CP) that selectively stains RNA in nucleoli of living cells is prepared. CP shows a deep-red emission (658 nm) and a large Stokes shift because of the introduction of rigid-conjugated coumarin moiety into the molecular structure. Cell imaging experiments indicate that CP can rapidly stain nucleoli in living cells by binding with nucleolar RNA, showing performance superior to commercially available nucleoli dye SYTO RNASelect in terms of high photostability and selectivity. More significantly, these excellent properties together with low cytotoxicity enable CP to monitor nucleolar RNA changes during mitosis, and after treating with anti-cancer drugs cisplatin, actinomycin D and α-amanitin. Thus, CP could be a potential tool for real-time, long-term visualization of the dynamic changes for nucleolar RNA and evaluation of the therapeutic effect for anticancer drugs that targeted RNA polymerase I (Pol I). & 2014 Elsevier B.V. All rights reserved.
Keywords: RNA Nucleoli Fluorescent imaging Real-time
1. Introduction RNA molecules are responsible for a wide range of functions in living cells, from physical conveyance and interpretation of genetic information, structural support for molecular machines, and regulation/silencing of gene expression, to essential catalytic roles (Prasanth and Spector, 2007; Storz et al., 2005). The ability to acquire complete spatial–temporal profiles of RNA synthesis, processing, and transport is therefore critical to understand cell function and behavior in health and disease conditions (Bao et al., 2009). In addition, most of the RNA in the nucleus is localized to the nucleolus, where is the key site of ribosomal RNA (rRNA) transcription, processing, and ribosome assembly (Yusupov et al., 2001). As one of the most powerful techniques for monitoring bimolecular in living systems, fluorescence imaging techniques are widely used to visualize morphological details of RNAs in cells. Those include microinjection of fluorescently tagged RNA molecules (Mhlanga et al., 2005; Wilkie and Davis, 2001), fluorescence in situ hybridization (Guo et al., 2012; Simon et al., 2010; Volpi and Bridger, 2008), molecular beacons (Chen and Tsourkas, 2009), and small-molecule fluorescent probes (Li and Chang, 2006; Liu et al., n
Corresponding author. E-mail address: [email protected] (P. Wang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.12.055 0956-5663/& 2014 Elsevier B.V. All rights reserved.
2011; O’Connor et al., 2009). Among these, small-molecule fluorescent probes are the most practical tool for live cell imaging, because the good cellular permeability make it does not require fixation of cells which can disturb the cell membrane and organelles. Considerable efforts have been exerted to develop small-molecule fluorescent probes for RNA imaging in living cells. However, in comparison with DNA probes, RNA probes, especially nucleolar RNA probes for live cell imaging are quite rare due to better affinity for DNA and poor nuclear membrane permeability. In 2006, Chang et al. first reported three selective RNA-binding styryl probes screened from 88 compounds through RNA assay and live cell imaging methods (Li and Chang, 2006). Recently, Song and Liu et al. also find other small-molecule probes for RNA in nucleoli and cytoplasm based on styryl dyes (Liu et al., 2014; Song et al., 2014). In addition, Stevens et al. designed an energy transfer probe for duplex RNA in cellular imaging (Stevens et al., 2008). Peng et al. reported a near-infrared RNA fluorescent probe for imaging living cells assisted by the macrocyclic molecule CB7 (Li et al., 2013). So far, the only commercially available nucleolar RNA probe is a green fluorescent dye known as “SYTO RNASelect”, whose molecular structure has not been published to date. However, these reports on nucleolar RNA staining also have problems of poor photostability, high cytotoxicity, short emission wavelength (o 600 nm), or poor cell permeability. Thus it is highly desirable to develop
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new fluorescent probes with excellent performance to monitor the changes of nucleolar RNA. The nucleolus is a subnuclear organelle that contains ribosomal RNA gene clusters and ribosome biogenesis factors. In this study, a fluorescent probe CP was development by rigid hybridizing coumarin and pyronin moieties to form crescent-shape structure. The intramolecular charge transfer reservation and planarity amplification resulted in large Stokes shift and deep red emission. Then CP was found to be a potential fluorescent probes for imaging RNA distribution within the nucleolar. The photophysics properties of CP and its interaction with nucleic acid (NA) have been investigated through absorption spectroscopy and fluorescence spectroscopy. Moreover, we successfully used CP to study the details of nucleolar RNA dynamic changes during mitosis, as well as after treatment with inhibitor of RNA polymerase I (Pol I).
2. Experimental section 2.1. Materials and apparatus All commercial chemicals were used without further purification. 7-amino-1-methyl-1,2,3,4-tetrahydroquinoline hydrochloride was purchased from HangZhou Trylead Chemical Technology Co., Ltd. Escherichia coli total RNA and Calf thymus (CT) DNA were obtained from Sigma-Aldrich. The stock solutions of CP were prepared in DMSO (1.0 10 2 M). All UV–vis and fluorescence spectra in this work were recorded using Hitachi U3900 and Hitachi F4600 fluorescence spectrometers, respectively. All measurements were carried out at room temperature. Water was purified by a Millipore filtration system. 1H NMR (400 MHz) and 13 C NMR (101 MHz) spectra were collected using a Bruker Advance 400 spectrometer with tetramethylsilane as an internal standard. Electrospray ionization high resolution mass spectra (ESI-HRMS) were obtained usi a Bruker Apex IV FTMS. Fluorescent images were acquired with a Nikon C1si laser scanning confocal microscope, equipped with a live-cell incubation chamber maintaining a humidified atmosphere of 37 °C and 5% CO2. Images were processed by NIS-element analysis AR 4.13.00. Clear nucleoli of Hela cells amplification imaging were obtained by a Delta Vision OMX imaging system.
2.2. Synthesis CP can be readily prepared by condensation reaction of 3-aldehyde-4-chlorocoumarin derivates and 3-aminophenol. Detailed synthetic routes and methods are shown in Scheme 1 and Supplementary information. CP: 1H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H), 8.01 (d, J¼ 9.5 Hz, 1H), 7.74 (s, 1H), 7.35 (d, J ¼9.3 Hz, 1H), 7.23 (s, 1H), 6.68 (s, 1H), 3.55 (s, 2H), 3.36 (s, 6H), 3.16 (s, 3H), 2.81 (s, 2H), 1.93 (d, J ¼10.8 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 161.61, 158.36, 158.14, 157.00, 156.54, 154.15, 145.09, 133.80, 122.81, 122.63, 116.27, 115.74, 105.39, 99.56, 97.34, 96.46, 50.97, 40.96, 26.34, 20.44. ESI-HRMS m/z calcd for [C22H21N2O3] þ 361.15467, found 361.15508. 2.3. Determination of quantum yield Fluorescence quantum yields (ΦU) are measured using cresyl violet in methanol as the reference (ΦR ¼ 0.54) based on the following equation:
⎛ A ⎞⎛ n 2 ⎞ ΦU = ΦR⎜ U ⎟⎜⎜ U2 ⎟⎟ ⎝ AR ⎠⎝ nR ⎠ where AU and AR are the integrated areas under the corrected fluorescence spectra for the sample and reference, nU and nR are the refractive indexes of the sample and reference, respectively (Magde et al., 1979). 2.4. Cell culture and staining Hela, A549 and L929 were gifted from the center of cells, Peking Union Medical College. L929, A549 or Hela cells were grown in RPM1640, McCoy's 5A or Dulbecco's modified Eagle's medium (DMEM) respectively, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated under 5% CO2 at 37 °C. Cells were seeded on confocal dish for imaging 12–24 h prior to conduction of experiment. Below is a general procedure for labeling living cells. Cells were adhering to coverslips. CP was dissolved in DMSO to a final concentration. After added the appropriate of dye stock solution directly to the culture medium, cells were incubated for 20 min at 37 °C in 5% CO2, and then imaged in dye medium at 37 °C in 5% CO2 without wash. For colocalization experiments, CP was
Scheme 1. Synthesis of CP. Conditions: (a) 85% phosphoric acid, reflux, 24 h; (b) diphenyl malonate, toluene, reflux, 6 h; (c) phosphoroxychloride, 50 °C, 30 min; and (d) 3aminophenol, acetic acid, 85 °C, 2 h, perchloric acid .
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incubated with Hela cells in DMEM for 20 min, and then the medium were replaced with fresh medium in the presence of SYTO RNASelect for 20 min. The cover slips were washed by PBS twice and imaged.
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of fresh medium containing 5 mM CP. After incubated at 37 °C, 5% CO2 for 30 min, the dish was placed on the microscope platform with a live-cell incubation chamber. Fluorescence images at Cy5 channel were acquired every 4 min to capture the dynamic process of nucleoli in cells undergoing cell division.
2.5. DNase and RNase treatment 2.9. Drug treatment Hela cells were fixed in pre-chilled methanol at 20 °C for 1 min. The cell membrane was permeablized with 1% Triton X-100 in PBS for 1 min at room temperature. After rinsing with PBS twice, 0.5 μL 10 mM CP stock solution and 1 mL PBS solution were added. Cells were then incubated in this dye PBS solution for 20 min at room temperature before being rinsed by clean PBS twice. A total of 1 mL clean PBS (as control experiment), 20 u/mL DNase (Takara), or 25 mg/mL DNase-Free RNase (Takara) were added into the three other dishes and incubated at 37 °C in 5% CO2 for 2 h. Cells were rinsed again by clean PBS twice before imaging. The fluorescent imaging pictures were obtained by using an equal exposure time for control, DNase, and RNase experiments.
Hela cells were seeded in 35 mm dishes and grown until they were 75–80% confluent. Culture medium was replaced by 2 mL of fresh medium containing 5 mM CP. After incubated at 37 °C, 5% CO2 for 30 min, the cells were treated with anticancer drugs (cisplatin (40 mM), actinomycin D (0.05 mg/mL) or α-amanitin (10 mg/mL)). The dish was placed on the microscope platform with a live-cell incubation chamber during imaging processes. Fluorescence images at Cy5 channel were acquired every 20 min, lasting 7 h (cisplatin), 6 h (actinomycin D), 3 h (α-amanitin), respectively.
3. Results and discussion 2.6. Cytotoxicity test 3.1. Synthesis and characterization of CP CP stock solutions were diluted by fresh medium into three desired concentration (1, 3, 5, 7, 10 μM). Hela cells were cultured in a 96-well plate for 12–24 h before the conduction of the experiments. The cell medium was then exchanged by different concentrations of CP medium solutions. They were then incubated at 37 °C in 5% CO2 for 24 h before cell viability was measured by the MTT assay. The cell medium solutions were exchanged by 100 μL of fresh medium, followed by the addition of 20 μL (5 mg/mL) MTT solution to each well. The cell plates were then incubated at 37 °C in 5% CO2 for 4 h. Absorbance was measured at 570 nm. The absorbance measured for an untreated cell population under the same experimental conditions was used as the reference point to establish 100% cell viability. Duplicated experiments have been tested. 2.7. Photostability test Hela cells were incubated in 1 mL culture solution consist of 5 μM CP or 1 μM RNASelect for 20 min and imaged by confocal microscope (Nikon C1si) without wash. The laser powers 488 nm and 640 nm were used to irradiate the RNASelect and CP stained cells respectively and the fluorescence were collected at FITC channel for RNASelect and Cy5 for CP by no delay scan mode. The initial intensity referred to the first scan of each dye. 2.8. Nucleolar dynamic observations during mitosis Hela cells were seeded in 35 mm dishes and grown until they were 75% to 80% confluent. Culture medium was replaced by 2 mL
The crescent-shape probe CP can be readily prepared by condensation reaction of 3-aldehyde-4-chlorocoumarin derivates and 3-aminophenol. Detailed synthetic routes and methods are shown in Scheme 1. Compound 2 was acquired by interesterification and Friedel–Crafts reaction of compound 1 with diphenyl malonate, whereas compound 3 was synthesized by Vilsmeier–Haack reaction. Eventually, CP was obtained from the condensation reaction of compound 3 with 3-aminophenol. The overall chemical structure of CP was confirmed via 1H NMR, 13C NMR, and ESI-HRMS (Fig. S1). 3.2. Photophysics properties Absorption and fluorescence spectra of CP in CH2Cl2 and Tris– HCl–EDTA (TE) buffer solution (pH ¼7.4) were investigated (Fig. 1). Corresponding photophysical data are summarized in Table S1. The maximum absorption of CP is 598 nm, and the maximum emission is 658 nm in TE buffer solution. Larger Stokes shift (60 nm) of CP in TE buffer solution is observed compared with that of xanthene in aqueous solution, which could reduce self-absorption, as well as obtain high-resolution and low-detection limits. Fluorescence quantum yield of CP is 0.22 in CH2Cl2 and only 0.01 in TE buffer solution (Table S1). The striking contrast of fluorescence quantum yields in different polar solvents indicating CP is sensitive to environments. In addition, the pH effect (5–8) of CP was also investigated (Fig. S2), and sensitivity to acid conditions was observed for CP. This may attribute to the containing of a
Fig. 1. Photophysical properties of CP. (A) Absorption and (B) fluorescence spectra of CP (10 μM) in CH2Cl2 and Tris–HCl–EDTA (TE) buffer solution (pH ¼7.4).
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Fig. 2. Fluorescent images of living cells by CP. (A) CP (5 μM) was incubated with Hela cells in Dulbecco's modified Eagle's medium (DMEM) for 20 min and imaged. (B) Fluorescence intensity profile of selected region in (A). (C) Clear nucleoli amplification imaging of Hela cells with 5 μM CP by Delta Vision OMX system. (D) Fluorescence intensity profile of selected region in (C). (E) A549 cells incubated with 5 μM CP for 20 min in McCoy's 5A and imaged. (F) L929 cells incubated with 5 μM CP for 20 min in RPM1640 and imaged. Images excited at 640 nm laser power, Cy5 channel.
positive charge in molecular structure (Arden–Jacob et al., 2001; Wang et al., 2010). 3.3. Fluorescent imaging properties in living cells To examine the cellular permeability/uptake, laser confocal imaging studies of CP were carried out in different cells, including tumor cells (HeLa and A549 cells) and normal cells (L929 cells) (Fig. 2). A red semiconductor laser (640 nm) was used as the light source for cell imaging and this could avoid cellular autofluorescence interference. After incubation with CP (5 μM) for 20 min, a clear nucleolar staining in the nucleus and some bright spots in the cytoplasm were observed for CP (Fig. 2A). No obvious cell morphology or viability change was found during the imaging. The fluorescence intensity in the nucleoli is about 30 times higher than that in the nucleus (Fig. 2B), and high signal/noise ratio achieved a strong contrast for simultaneous visualization of the nucleus and nucleolus. A clear nucleolar amplification imaging was also obtained by the Delta Vision OMX imaging system. Nucleolar fluorescence exhibited a honeycomb structure with different apertures and micro-holes (Fig. 2C). Variational intensity in region holes also indicated uneven distribution of CP in the nucleoli (Fig. 2D). Similar staining is observed in A549 and L929 cells (Fig. 2E and F), indicating that CP has good nuclear membrane permeability in cancer and normal cells. 3.4. Cell localization and the interaction with NA A commercially available nucleolar stain SYTO RNASelect was used to co-stain cells with CP to confirm that the observed bright
spots in the nucleus were related to the fluorescence of CP that localized at the nucleoli. As we know, the nucleolus contains abundant proteins and RNAs, and SYTO RNASelect stained RNA in nucleoli. Orange fluorescence indicated the same subcellular localization of the nucleoli in SYTO RNASelect and CP (Fig. 3A). The fluorescence throughout the cytoplasm was predominantly associated with lysosome by co-staining with LysoTracker Green (Fig. S3). The brighter fluorescence of CP in lysosome may result from acidic environment with the lysosomal pH. This might be attributed to lysosomes are acidic organelles. In addition, deoxyribonuclease (DNase) and ribonuclease (RNase) digest experiments were also performed to identify the stained species by CP in the nucleoli. DAPI and SYTO RNASelect were tested as controls. Upon treatment with DNase (Fig. 3B), no significant loss of fluorescence in the nucleoli occurred for CP. By contrast, after RNase digestion, the nucleolar fluorescence signals of CP were completely lost and redistributed in the nucleus. CP exhibited a similar behavior in the digest experiment with SYTO RNASelect, indicating that nucleolar fluorescence originated from the binding of CP with RNA in the nucleoli. To further investigate the CP's preference to RNA in vitro, absorption and fluorescence spectra of CP upon nucleic acid (NA) binding were investigated in buffer solution (Fig. 4 and Table S2). As Fig. 4A shows, upon addition of NA at a 100:1 CNA/CCP ratio, a pronounced bathochromic effect in absorption spectra (above 20 nm) is observed for CP, which reveal the strong interaction between CP and NA. The fluorescence intensity of CP upon RNA displays a larger fluorescence enhancement than DNA for the same amount of NA (Fig. 4B). The result might be attributable to crescent-shape ligand with positive charges may prefer binding with
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Fig. 3. Cell Localization of CP. (A) Co-localization of SYTO RNASelect (1 μM). (B) DNase and RNase digest experiments. Images of CP (0.5 μM), as well as DAPI (1 mg/ml) and SYTO RNASelect (0.5 mM), are shown as comparison experiments. Equal exposure was used for the same dye imaging. DAPI (blue: DAPI channel), SYTO RNASelect (green: FITC channel), and CP (red: Cy5 channel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Spectra changes of CP in the present of NA. (A) Absorption and (B) fluorescence spectra of CP (10 μM) in TE buffer (pH¼ 7.4) at 100:1 CNA/CCP ratio.
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RNA (Cetinkol and Hud, 2009; Sinha and Kumar, 2009). A similar phenomenon for SYTO RNA-Select was also observed in buffer solution (Fig. S4), indicating that CP and SYTO RNA-Select both have higher response to RNA than to DNA in solution. However, CP showed a better selectivity for RNA in nucleus in living cell experiments, which may be possibly due to easy accumulation in nucleolus for CP and the difference between structure of NA in solution and their real state in cells (Li and Chang, 2006; Song et al., 2014). Additionally, the selectivity of CP for RNA was evaluated by the fluorescence intensity response in TE buffer solution. As illustrated in Fig. S5, the other biological molecules including Lcysteine, histidine, glucose, adenosine triphosphate (ATP), Mg2 þ , and Cl do not give rise to significant increase in fluorescence intensity at 658 nm. NA is important biological macromolecules that function in encoding, transmitting, and expressing genetic information. The simultaneous visualization of DNA and RNA in nucleus is important using different dyes during the cell imaging. We investigated the counterstaining behavior for CP with DNA dye Hoechst33342 (Fig. 5). The blue nuclear stain in the DAPI channel and the red nucleolar stain in the Cy5 channel can be clearly visualized. The result also indicates that CP has excellent counterstaining compatibility with Hoechst33342. 3.5. Cytotoxicity and photostability Cytotoxicity and photostability are two important factors for evaluating applicability of new fluorescent probes in live cell imaging. The cytotoxicity of CP toward HeLa cell lines was studied using standard MTT assay after 24 h of incubation (Fig. 6A). CP
exhibited good cell tolerability at imaging concentration after 24 h of incubation. The photostability of CP and SYTO RNASelect in cell imaging were also examined. CP exhibited better photostability than SYTO RNASelect. The fluorescence signals of SYTO RNA-Select decreased by 80% after 52 scans (Fig. 6B). By contrast, no significant decrease in intensity was observed under continuous irradiation for 300 scans in 10 min, indicating that CP is a promising dye for long-term nucleolar RNA imaging. 3.6. Dynamic changes of nucleolar RNA during mitosis Cells have to double in size before dividing, and cell growth correlates with rRNA synthesis. In the cell cycle, the nucleolus is assembled at the end of mitosis and throughout interphase, and disassembles in prophase. Transcription is silenced during mitosis, gradually increases during G1-phase, and reaches maximal levels at S- and G2-phase (Russell and Zomerdijk, 2006). Nucleolar assembly of the rRNA processing study in living cells usually utilize green fluorescence protein-tagged nucleolar proteins and linear oligonucleotide probe (Molenaar et al., 2001; Savino et al., 2001). However, no small organic probe was used in monitoring the dynamic process of the RNAs in the nucleolus during mitosis. With good cell permeability, good photostability, and low cytotoxicity, CP was successfully used in monitoring the temporal and spatial changes of the nucleolar RNA in Hela cells during mitosis (Movie S1). Cells were stained with CP and imaged with timelapse. Fig. 7 shows a 7 h laser scaning confocal experiment that was recorded through a 60x objective in a cell preceded through mitosis. Fluorescence is clearly observed in two large size nucleoli at interphase (Fig. 7A–C), indicating high transcriptional activity in the
Fig. 5. Live cell counterstain CP (5 μM) with Hoechst33342 (1 μM). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 6. Cytotoxicity and photostability of CP. (a) Cytotoxicity of CP; (b) photostability of CP (5 μM) and SYTO RNASelect (1 μM) with increasing number of scans during cell imaging.
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Fig. 7. Dynamics of nucleoli of stained HeLa cells by CP during mitosis. (A–I) Time-dependent fluorescence imaging after addition of 5 mM CP.
Fig. 8. Time-lapse imaging of nucleolus RNA in HeLa cells with cisplatin, Act D, or α-amantin treated using CP (5 μM) in the range of hours.
nucleoli. After about three hours, the cell entered mitosis, rDNA transcription was repressed, and RNA synthesis ceased (Fig. 7C). In the next one hour, the nucleoli were distorted, reducing in size and disappearing, and no longer observed throughout mitosis, along with decreasing fluorescence in the nucleolar region and finally
fading away (Fig. 7D–G). During telophase, the nucleoli rapidly assemble due to the restoration of rDNA transcription and functional activity. As shown in Fig. 7, very small nucleoli were observed and increased in size along with daughter cells, concomitantly with increasing fluorescence intensity in the nucleoli.
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The mitosis time of HeLa cells is about 1.5 h. Then cell enter early interphase, which three or four nucleoli visualized in each nucleus (Yamauchi et al., 2007). A similar observation can also be obtained in normal L929 cells (Movie S2). The results further confirm that CP does not disturb cell proliferation and is expecting to be used in localization of nucleolar RNA dynamic process. Supplementary material related to this article can be found online at. http://dx.doi.org/10.1016/j.bios.2014.12.055 3.7. Dynamic changes of nucleolar RNA under treatment with anticancer drug As a potential tool for real-time, long-term monitoring of the dynamics of RNA in the nucleolus, an important application of CP is to study the mechanism of drug action and evaluate therapeutic effect. A new therapeutic approach to kill cancer cells is based on the specific inhibition of RNA polymerase I (Pol I) activity (Montanaro et al., 2013). Pol I is located in the nucleolus, and its sole function is the transcription of genes encoding rRNA, accounting for at least 60% of cell transcriptional activity. Almost all signaling pathways that affect cell growth and proliferation directly regulate rRNA synthesis, and their downstream effectors converge at the Pol I transcription machinery. The activity of Pol I is related to the synthesis rate and the amount of RNAs in the nucleolus. Real-time monitoring of RNA changes in the nucleolus during cell apoptosis caused by inhibited Pol I will provide valuable insights to drug mechanism and detection of therapeutic efficiency. Cisplatin (cis-diamminedichloroplatinum (II)) is the flagship compound for the treatment of a wide range of cancers. Targets for DNA and redistribution of RNA Pol I have important functions in the clinical success of cisplatin (Jordan and Carmo-Fonseca, 1998). We used CP to monitor nucleolar RNA changes induced by cisplatin. As Fig. 8 shows, no obvious changes in control experiments occurred (Movie S3). However, nucleolus was packaged into granules after 5 h in cisplatin-treated HeLa cells because of the redistribution of RNA Pol I (Fig. 8 and Movie S4). This result is well consistent with the silver staining method (Horky et al., 2001). Similarly, the decreased amount of nucleolar RNA resulted in weaker fluorescence in the nucleolus in the presence of actinomycin D (Act D) due to inhibition of RNA Pol I (Fig. 8 and Movie S5) (Hofgartner et al., 1979). Conversely, the use of α-amanitin, an inhibitor of RNA polymerase II and III which localize in nucleus and cytosol, resulted in no distinct changes of RNA amount in the nucleolus (Fig. 8 and Movie S6) (Rudd and Luse, 1996). The results further demonstrate that CP does target nucleolus RNA and can be used as an imaging tool to evaluate the therapeutic effect of Pol I targeted anticancer drugs.
4. Conclusion In summary, a crescent-shaped probe CP that contains coumarin and pyronin Y-fused conjugated skeleton was designed and synthesized. With good cell permeability, low cytotoxicity, and high photostability, CP can be successfully used in real-time monitoring of nucleolar RNA dynamics during mitosis and apoptosis induced by anticancer drugs that target RNA Pol I. These results provide useful guidelines for CP as a powerful tool for studying the dynamic changes of nucleolar RNA in response to
external stimuli, including anti-cancer drugs, stress, temperature, and so on.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 21373250, 61227008, and F040801). We would like to thank the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science for our Structured Illumination Microscopy work and we would be grateful to Shuoguo Li for her help of analyzing EM images.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.055.
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