Letters Cite This: ACS Chem. Biol. 2017, 12, 2546-2551
pubs.acs.org/acschemicalbiology
Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule Hidefumi Iwashita,*,†,‡ Satoru Torii,§ Noriyoshi Nagahora,‡ Munetaka Ishiyama,† Kosei Shioji,‡ Kazumi Sasamoto,† Shigeomi Shimizu,§ and Kentaro Okuma‡ †
Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kumamoto 861-2202, Japan Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-Ku, Fukuoka 814-0180, Japan § Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan ‡
S Supporting Information *
ABSTRACT: There has been a growing interest in mitophagy, mitochondria-selective autophagy, which plays an essential role in maintaining intracellular homeostasis. We have developed a smallmolecule fluorescent probe, Mtphagy Dye, for visualizing mitophagy, which was readily synthesized from a known perylene derivative, perylene-3,4-dicarboxylic anhydride. Mtphagy Dye has suitable fluorescent properties for detecting mitochondrial acidification during mitophagy in the long-wavelength region that does not damage mitochondria. Using Mtphagy Dye, we were able to visualize mitophagy with both cases of Parkin-dependent and -independent HeLa cells.
A
reported. Zhang et al., for example, reported real-time monitoring of mitophagy with a tetraphenylethene derivative, TPE-Py-NCS, that has an aggregation-induced type of fluorescent moiety along with an isothiocyanate group to be immobilized within mitochondria through covalent bonding.11 Lee et al. also described a unique approach for detecting mitochondrial acidification by using a pH-sensitive fluorescent probe composed of a naphthalimide moiety as a fluorophore.12 Both probes, however, have their excitation maximum at around 400 nm, which is much shorter than the wavelength for laser excitation at 488 nm, requiring the use of higher probe concentrations and/or higher laser output in fluorescence microscopy. In fact, high concentrations were employed with these probes in fluorescence monitoring of mitophagy, in which the intact mitochondria might have been damaged by enhanced depolarization through the use of excess concentrations. We therefore think it very important for live cells to employ a lower probe concentration that does not impair the mitochondrial functions. Here, we report the synthesis of a novel fluorescent small molecule, Mtphagy Dye (Scheme 1), and its applications to visualize mitophagy. Mtphagy Dye contains a perylene-3,4dicarboximide moiety as a highly sensitive fluorescent group as well as a triphenylphosphonium moiety as a mitochondria localizing group. In addition, chloromethyl and piperazine
utophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles.1−4 The damaged cellular components are sequestered into double membrane vesicles, called autophagosome, which are then fused to lysosome, and the sequestered cargo molecules are degraded by digestion. In particular, mitophagy, mitochondria-selective autophagy, serves as a specific elimination system of dysfunctional mitochondria, and it may play critical roles in aging as well as neurodegenerative diseases, such as Parkinson’s disease, that are caused by the accumulation of depolarized mitochondria.5−7 Thus, mitophagy plays a central role in maintaining intracellular homeostasis. Although the pH-sensitive fluorescent protein, Keima, is widely used for visualizing mitophagy, it requires a plasmid transfection technique to be expressed.8−10 Keima expressed in mitochondria can be excited at 440 nm at neutral pH. When the pH becomes acidic during mitophagy, the excitation wavelength shifts to 580 nm, which enables the visualization. Among several approaches reported so far with fluorescent proteins, Keima is the only available technique to visualize mitophagy in live cells. The application, however, is limited to certain types of cells because of the requirement of transfection techniques. We therefore set out to develop a sensitive technique for visualizing mitophagy with a small-molecule fluorescent probe that can be applied to any types of cells and also is useful, for example, for drug screening with patientderived iPS cells. As analogous approaches, several methods for mitophagy detection using fluorescent small molecules have been recently © 2017 American Chemical Society
Received: July 31, 2017 Accepted: September 19, 2017 Published: September 19, 2017 2546
DOI: 10.1021/acschembio.7b00647 ACS Chem. Biol. 2017, 12, 2546−2551
Letters
ACS Chemical Biology Scheme 1. Synthetic Route to Mtphagy Dyea
(a) β-alanine benzyl ester, Zn(CH3CO2)2, quinoline, 120 °C, 16 h; (b) 10% Pd/C, H 2 , THF/EtOH; (c) (2-aminoethyl)triphenylphosphonium bromide, DMT-MM, DIEA, DMF, r.t., 2 h; (d) Br2, K2CO3, 1,2-dichloroethane, 100 °C, 2 h; (e) piperazine, 2methoxy ethanol, 130 °C, 16 h; (f) α,α′-dichloro-p-xylene, K2CO3, CH3CN, 100 °C, 16 h. a
Figure 2. Fluorescence enhancement of Mtphagy Dye at acidic pH with isolated mitochondria. (A) Fluorescence images costained with MitoTracker at pH 4.0 and pH 7.4. (B) Quantification data of red fluorescence of Mtphagy Dye, **P < 0.01. (C) Flow cytometric analysis under conditions at pH 4.0, pH 7.4, and pH 4.0 with 0.01% Triton.
and to serve as a pH sensor for mitochondrial acidification, respectively. Mtphagy Dye shows weak fluorescence under neutral conditions such as in cytosol but becomes highly fluorescent under acidic conditions such as in lysosome due to protonation of the piperazine moiety, which cancels out the fluorescence quenching by this moiety. Using Mtphagy Dye at a low concentration under physiological conditions, we were able to monitor mitophagy with Parkin-expressed HeLa cells which was induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, as well as Parkin-independent mitophagy triggered by anticancer drugs. Unlike fluorescent protein-based probes, Mtphagy Dye readily detected mitophagy by simply adding the dye solution to the cells. Mtphagy Dye was synthesized in overall six steps in a moderate yield from perylene-3,4-dicarboxylic anhydride (1),13,14 as outlined in Scheme 1. Briefly, condensation of βalanine benzyl ester with compound 1 in the presence of zinc acetate gave ester 2, which was then deprotected to yield acid 3. To acid 3 was conjugated a mitochondria-localizing group15 using a coupling reagent to yield compound 4. The piperazine moiety was introduced via bromination to compound 4, giving precursor 6. Further introduction of the chloromethyl unit with xylylene chloride finally afforded Mtphagy Dye. Figure 1A shows the excitation and emission spectra of Mtphagy Dye measured at pH 4.0 (orange) and 7.4 (blue) in a buffer solution containing 50% acetonitrile. As expected, Mtphagy Dye was found to have much longer fluorescence wavelengths compared to TPE-Py-NCS11 or naphthalimidetype fluorescent probe,12 with the single emission peak at 700 nm when excited at 520 nm (pH 4.0) or at 720 nm when excited at 530 nm (pH 7.4), in addition to a large Stokes shift of roughly 170 nm (Table S1). These fluorescence properties
Figure 1. Fluorescence properties of Mtphagy Dye. (A) Excitation (---) and emission (―) spectra of Mtphagy Dye at pH 4.0 (orange) and pH 7.4 (blue) in 50% (v/v) aqueous acetonitrile. (B) pHdependent changes of the fluorescence intensity of Mtphagy Dye.
groups are also incorporated within the molecule to be immobilized to mitochondrial proteins through thiol groups 2547
DOI: 10.1021/acschembio.7b00647 ACS Chem. Biol. 2017, 12, 2546−2551
Letters
ACS Chemical Biology
Figure 3. Parkin-dependent mitophagy induced by CCCP (10 μM) with Mtphagy Dye (0.1 μM). (A) Confocal microscopic images of normal and Parkin-expressed HeLa cells with or without bafilomycin A1 (baf) co-stained with MitoTracker (0.1 μM). Arrows: overlapped puncta. (B) Quantification data of A; **P < 0.01. Red lines indicate the mean values. (C) Confocal microscopic images of GFP-LC3 expressed normal HeLa cells (Parkin (−)) and GFP-LC3 and Parkin co-expressed HeLa cells (Parkin (+)) with or without bafilomycin A1 (baf). Arrowheads: GFP-LC3 alone puncta. (D) Quantification data of C; **P < 0.01. Red lines indicate the mean values. Scale bar: 10 μm.
moiety, which is no longer effective at acidic pH and thus allows us to monitor acidification during mitophagy. In order to make sure whether this fluorescence quenching is actually due to the PeT mechanism, theoretical calculations were carried out. The theoretical investigation showed that both the HOMO and LUMO of the dye predominantly delocalized on π-electrons of the perylene-3,4-dicarboximide moiety, whereas the HOMO−1 exclusively contained lone-pair electrons of the nitrogen atom of the piperazine ring (Figure S3). These results suggest that low-energy singlet excitations of Mtphagy Dye could be allowed for π−π* (HOMO to LUMO) and, to some extent, n−π* (HOMO−1 to LUMO) electron transitions and that the n−π* transition corresponds to a charge transfer character. The fluorescence quenching, therefore, can be explained more likely by a charge transfer rather
are suitable for widely used confocal laser microscopy and would allow monitoring of mitophagy with a high sensitivity at a longer wavelength in the near-infrared region where there is much less endogenous fluorescence background. In addition, the fluorescent intensity of the dye in aprotic solvents such as DMSO or acetonitrile is higher than that in protic solvents such as methanol (Figure S1); it also decreases with increasing water content in aqueous DMSO solution (Figure S2). The pKa value was determined to be 6.0 for Mtphagy Dye by using the Henderson−Hasselbalch equation based on a plot of the fluorescence intensity at 700 nm versus various pH, being smaller than that of the reported naphthalimide-type probe (Figure 1B). This fluorescence-pH profile might be explained by a photoinduced electron transfer (PeT) mechanism from the nitrogen atom of the piperazine ring to the peryleneimide 2548
DOI: 10.1021/acschembio.7b00647 ACS Chem. Biol. 2017, 12, 2546−2551
Letters
ACS Chemical Biology
Figure 4. Parkin-independent mitophagy induced by etoposide (10 μM) or rapamycin (1 μM) with Mtphagy Dye (0.1 μM). (A) Confocal microscopic images of normal HeLa cells co-stained with MitoTracker (0.1 μM). Arrows: overlapped puncta. (B) Quantification data of A; **P < 0.01. Red lines indicate the mean values. (C) Confocal microscopic images of GFP-LC3 expressed HeLa cells. Arrowheads: GFP-LC3 alone puncta. (D) Quantification data of C; **P < 0.01. Red lines indicate the mean values. Scale bar: 5 μm.
Also with the isolated mitochondria stained with Mtphagy Dye, we next examined immobilizing effects of the dye under membrane-depolarized conditions. CCCP, an uncoupling reagent for mitochondrial membrane potential, was used at 3 μM or 10 μM to depolarize the mitochondrial membrane. Double staining experiments with MitoTracker Green at pH 4.0 revealed that Mtphagy Dye was retained within the mitochondria in the presence of CCCP without any apparent leakage, possibly owing to the anchoring effect of the chloromethyl group of the dye (Figure S5A and B). Similar results were also obtained with flow cytometry (Figure S5C). The fluorescence enhancement of Mtphagy Dye upon acidification was also observed with live HeLa cells, the pH of which was equilibrated at 4.5 using a high-concentration potassium buffer containing nigericin16 or at 7.4 with HEPES buffer. Although the fluorescence was hardly observed at pH 7.4 with the dye concentration at 0.1 μM, it was much brighter at pH 4.5 with the same dye concentration. In both cases, the dye seemed to localize in mitochondria, as observed with fluorescence images stained with MitoTracker (Figure S6). It was therefore expected that the dye was likely to respond to acidification during mitophagy with the large fluorescence enhancement.
than the PeT mechanism, for which further studies will be needed. In either case, these unique fluorescence properties of the dye probably stem from the π-system of the perylene-3,4dicarboximide moiety to which the piperazine ring is adjacent. The observation that Mtphagy Dye emits intense fluorescence in hydrophobic media but the fluorescence is suppressed in aqueous environments suggests that the dye is likely to exhibit very low background fluorescence when diffused into the cytoplasm of cells. We also checked the cytotoxicity of Mtphagy Dye and found that the dye was not cytotoxic at least up to 1.0 μM, the concentration we used in the following experiments (Figure S4). We first tested whether Mtphagy Dye was incorporated into isolated mitochondria and its fluorescence was enhanced at acidic pH. When isolated mitochondria were examined using fluorescence microscopy, Mtphagy Dye was very weakly observed in isolated mitochondria at pH 7.4, and the fluorescence was largely enhanced at pH 4.0 (Figure 2A and B). Consistent results were observed when fluorescence was measured using a flow cytometer (Figure 2C). Note that the fluorescence was largely diminished by the addition of Triton X-100 even at pH 4.0, suggesting that the dye is incorporated into the mitochondria. 2549
DOI: 10.1021/acschembio.7b00647 ACS Chem. Biol. 2017, 12, 2546−2551
Letters
ACS Chemical Biology
tool with good sensitivity and photostability for detecting mitophagic phenomena with a wide range of cell lines, particularly under conditions that do not impair mitochondrial functions, which is in sharp contrast to previously reported small-molecule fluorescent probes.
In order to further validate the efficiency of Mtphagy Dye in visualizing mitophagy, we performed an experiment to induce mitophagy by CCCP treatment with Parkin-expressed HeLa cells that had been produced by transfection of a Parkin plasmid vector. Parkin is an E3 ligase enzyme encoded by the PARK2 gene in human, a causative gene of Parkinson’s disease, and is known to mediate ubiquitin signaling to promote mitophagy.17 As expected, Mtphagy Dye showed very weak fluorescence with normal HeLa cells (Parkin (−)) that were not supposed to be responsive to CCCP (Figure 3A). On the other hand, red-colored fluorescent puncta due to the presence of Mtphagy Dye were observed with Parkin-expressed HeLa cells (Parkin (+) in Figure 3A). Red fluorescent puncta overlapped with those of MitoTracker, indicating that the dye accumulated in mitochondria showed fluorescence with decreasing pH during mitophagy when mitochondria fused with lysosome. This was also confirmed by the observation that the fluorescence was largely suppressed by the addition of bafilomycin A1 that is known to block the acidification, thereby inhibiting mitophagy (Figure 3A and B). Furthermore, GFPLC3, which is an autophagosome marker, was expressed in Parkin-expressed as well as normal HeLa cells, and then mitophagy was induced by CCCP in these cells (Figure 3C and D). Although weak fluorescence was observed in GFP-LC3expressed normal HeLa cells, the red fluorescence of Mtphagy Dye was nearly quenched. Under mitophagy-induced conditions, on the other hand, there were several green fluorescent puncta observed in GFP-LC3-expressed cells due to the formation of an autophagosome. The fluorescence brightness of GFP-LC3 is normally decreased when, in the late phase of autophagy, the autophagosome is fused with lysosome to form autolysosome whose pH is acidic. Mtphagy Dye, by contrast, enhances its fluorescence at acidic autolysosomal pH, which can be seen in several overlapping as well as non-overlapping fluorescent puncta in Figure 3C. When mitophagy was impaired by bafilomycin A1, Mtphagy Dye did not exhibit the fluorescence, whereas GFP-LC3 expressed cells showed even brighter fluorescence due to the accumulation of autophagosomes. These results support the idea that Mtphagy Dye localizes in mitochondria, which is recruited to the autophagosome upon the induction of mitophagy, as shown by GFP-LC3, and then is fused with lysosome resulting in fluorescence enhancement at acidic pH. Figure 4 shows that two types of autophagy inducers, etoposide18,19 as a DNA damaging anticancer drug and rapamycin20 as a cellular starvation mimic, have their ability to induce mitophagy in HeLa cells stained with Mtphagy Dye. Double staining with MitoTracker revealed that, in both cases of etoposide and rapamycin, red fluorescent puncta originated from mitochondria (Figure 4A and B). GFP-LC3 puncta formation was upregulated by both treatments (Figure 4C and D). Corresponding to the results of CCCP treatment, red fluorescence of Mtphagy Dye was observed in overlapping as well as non-overlapping fluorescent puncta with GFP-LC3 (Figure 4C and D). Taken together, Mtphagy dye was found to be also effective in detecting Parkin-independent mitophagy, which will be useful as a tool to study mitophagy. In summary, as a reliable visualizing technique of mitophagy, we have developed the mitochondria-selective pH-sensitive fluorescent dye, Mtphagy Dye, which localizes in mitochondria and emits strong fluorescence in the longer-wavelength region upon acidification during mitophagy. This method using Mtphagy Dye, therefore, can be a reliable and convenient
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00647. Details of reagents and instruments, synthesis of Mtphagy dye, experimental procedure, and supplementary data including figures, tables, and fluorescence images (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hidefumi Iwashita: 0000-0001-6709-0897 Noriyoshi Nagahora: 0000-0002-9663-0677 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Yuichiro Ueno, Dojindo Laboratories, for useful discussions and comments.
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