Subscriber access provided by JAMES COOK UNIVERSITY LIBRARY
Article
Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide Haihong He, Zhiwei Ye, Yi Xiao, Wei Yang, Xuhong Qian, and Youjun Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04510 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide Haihong Hea,†, Zhiwei Yeb,†, Yi Xiaob,*, Wei Yangb,*, Xuhong Qiana,*, Youjun Yanga,* a
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, China 200237 b
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Liaoning, China, 116024.
†
These authors contributed equally to this work.
ABSTRACT: Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease and aging. Molecular tools facilitating study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with sub-diffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.
Mitochondria undergo dynamic dislocation, morphological alteration, fusion and fission. These processes are vital for maintaining the physiological functions of mitochondria.1 Aberrations in mitochondrial dynamics are linked to mitophagy and cell survival.2,3 Nitric oxide (NO), an endogenous molecule implicated in vasodilation4, signal transduction5 and immune response6 etc. was recently recognized to also play regulatory roles on mitochondrial morphology and function.7-10 NO may activate PGC1α/NRF-1 to trigger biogenesis.11,12 NO can also inhibit Drp1 to induce excessive fission, which is further linked to neuronal injury and neurodegeneration.13-15 However, the plethora of mechanistic details of NO on mitochondrial dynamics remains largely unknown. It is noteworthy that studies on mitochondria dynamics have heavily relied on diffraction-limited fluorescence microscopies, for which the small diameter of mitochondria at ca. 250-500 nm represents a major challenge.16,17 Recent years have witnessed a paradigm shift toward the use of superresolution microscopic methods, e.g. stochastic optical reconstruction (STORM)18, photo-activated localization (PALM)19 and stimulated emission depletion (STED)20 in monitoring mitochondria dynamics21-24. Molecular tools facilitating convenient super-resolution monitoring of the synergy between nitric oxide and mitochondrial dynamics are anticipated. NO donors have often been employed alternatively to NO gas as the exogenous source in biological studies for convenience in handling.25 We expect to develop a NO donor, which is non-fluorescent and specifically localizes at mitochondria. Upon photo-activation, it releases both NO, which elicits downstream biological response, and a
fluorophore, which enables convenient monitoring of mitochondria dynamics. We further envisage that the fluorescence turn-on mechanism of this proposed NO donor is potentially compatible with the photo-activation localization microscopy (PALM) for super-resolution mapping of NO release if the two additional requirements are met. First, the NO donor does not absorb at where the resulting fluorophore absorbs to avoid erroneous activation by the excitation laser. Second, the resulting fluorophore should be bright and photostable so that enough photons are emitted to allow accurate localization before bleached. Regardless of the variety of NO donors reported in literature, such an NO donor is not yet available.26,27 Most release nitric oxide spontaneously without a photo-
Figure 1. The chemical structure and the ORTEP drawing of NOD550, and its photo-triggered activation and localization.
ACS Paragon Plus Environment
Analytical Chemistry triggering mechanism, such as NONOates, nitrosothiols, and metal-nitroso complexes. Among the rationally designed photo-triggered NO donors reported thus far,most does not yield a fluorescence turn-on upon NO release.2832 NOD545 is the first class of NO donors exhibiting a fluorescence turn-on upon photo-triggered NO release.33 However, the resulting naphthalimide fluorophore is not suitable for localization. Herein, we report a novel high-payload NO donor (NOD550), which enables mapping of NO release and simultaneous monitoring of the mitochondrial dynamics with super resolution localization microscopy.
1.00
A Emission λ ex= 520 nm
Absorption 0.75 0.50 0.25 0.00
300
400
500
600
Fluorescence Intensity (AU)
NOD550 was conveniently prepared via a two-step cascade starting from Rhodamine 6G in a high overall yield. The structure of NOD550 is established by NMR, MS and single-crystal X-ray diffraction (Figure 1). The two nitrosamine moieties are near perpendicular with respect to the xanthene moiety, with a dihedral angle of 83o, presumably due to the allylic strain induced by the presence of an ortho-methyl group. Also, it is interesting to note that both nitrosamine moieties are twisted to the same side of the xanthene plane, away from the lactone moiety.
Absorbance
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
7
1.0x10
B λex = 520 nm, λ em = 550 nm
6
7.5x10
at ca. 310 nm (ε = 7500 cm-1M-1) and does not absorb in the visible spectral region (blue line in Figure 2A). NOD550 is non-fluorescent. Irradiation of a NOD550 solution (10 µM) in phosphate buffer (50 mM at pH = 7.4) containing 5% DMSO as a co-solvent at 375 nm induced the appearance of an intense absorption band with a maximum at 520 nm. Excitation at 520 nm gave an emission band with a maximum at 550 nm, in agreement with the spectral properties of compound 1. The fluorescence enhanced essentially linearly in the first ca. 2500 s and then leveled off (Figure 2B). The step-wise photolysis mechanism of NOD550 to 1 via a mono-denitrosated intermediate (2) upon UV irradiation was confirmed by MS spectroscopy (Figure 2C). The formation of NO upon photolysis of NOD550 was also confirmed by EPR (Figure S2). High chemostability of NOD550 in biological milieu can avoid unintended fluorescence turn-on and is vital for localization microscopy. Mechanistically, denitrosation may occur via transnitrosation or reductive denitrosation. Therefore, NOD550 (10 µM) in phosphate buffer (50 mM at pH = 7.4) with 5% DMSO was incubated with various biological thiols, or biorelevant reducing agents, i.e. cysteine, glutathione, ascorbic acid, resveratrol, tryptophan, up to 10 mM. The absorption spectra of these solutions were unattenuated after 30 min (Figure S4).
6
5.0x10
6
2.5x10
0.0 0
700
1000
2000
3000
4000
5000
Time (s)
Wavelength (nm)
Figure 3. In vivo activation of NOD550 by 375 nm laser and subsequent bleaching of fluorophore 1 by 532 nm laser. The 2 375 nm laser (10 W/cm ) is time-gated while the 532 nm laser 2 (30 W/cm ) continuously on.
Figure 2. (A) The change of the UV-Vis absorption and emission spectra of NOD550 (10 µM) in neutral phosphate buffer containing 5% DMSO upon irradiation by 375 nm. (B) The fluorescence turn-on of NOD550 with respective to time. (C) MS data verifying the photolysis mechanism of NOD550.
Table 1. Spectral and photophysical parameters of NOD550 and the corresponding rhodamine fluorophore 1. Compound
λabs (nm)
λem (nm)
φ
ε (cm-1•M-1)
NOD550
310
n.d.
n.d.
7500
1
520
550
0.83
73900
Note: In phosphate buffer (50 mM, pH = 7.4) with 5% DMSO as a co-solvent. “n.d.” stands for not-detected.
The photo-triggered activation of NOD550 was studied with UV-Vis absorption and fluorescence spectroscopies. NOD550 has its maximal absorption wavelength located
The feasibility of photoactivation of NOD550 was evaluated in live cells. A green laser at 532 nm did not activate NOD550. Upon irradiation by a short pulse of UV activation laser (375 nm at 10 W/cm2), formation of ~240 molecules of fluorophore 1 was induced. Over continuous exposure to the green laser irradiation (532 nm at 30 W/cm2), these active fluorophores were bleached in ca. 20 s. This activation-bleaching cycle was repeated (for 13 times in Figure 3). Essentially the same number of fluorophores was activated by each pulse of 375 nm laser. This verifies that the 375 nm laser can efficiently and reliably photoactivate cellular NOD550 to release the parent fluorophore 1 in a time-gated fashion. The feasibility of PALM imaging with NOD550 in HeLa cells was studied with a total internal reflection fluorescence microscope (TIRFM). As the Z-axis location was at the edge of the TIRF illuminating field, the angle of the laser was slightly adjusted to a highly inclined situation following the variable-angle epifluorescence microscopy (VAEM)34,35 for better imaging the inside cell components. The imaging medium was RPMI 1640 culture media sup
ACS Paragon Plus Environment
Page 2 of 7
Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Figure 4. The utility of NOD550 for super resolution imaging mitochondria in live Hela cell. Wide-field image (a) and PALM image (b) of the mitochondria. (c) Enlarged map of the boxed area of image b showing individual release sites of fluorophore 1. (d) Transverse profile of two mitochondria along the yellow line in a and b. Fitting of the profile by two Gaussian functions (dashed line for a and solid line for b) gave the widths of two mitochondria. (e) Histograms of the number of photons per singlemolecule event. (f) Histograms of the localization precision. (g) The distance between fluorophore release sites along the yellow line cross the mitochondrion in image c. Scale bars: 3 μm (a and b); 500 nm (c).
plemented with 10% FBS, without either additional oxygen depletion system or reductant. The PALM image was constructed from 1500 frames (Figure 4b), collected during 10 s with a rate of 150 Hz. Compared to the relatively short imaging duration, a good localization precision of 19 nm was achieved (Figure 4f). The mean number of photons emitted from each fluorophore was estimated to be ~2800 (Figure 4e). Compared to the wide-field image (Figure 4a), PALM image gave much improved structural resolution and revealed detailed morphological information of a certain type sub-cellular organelle, the twists and crossovers of which suggested that they are likely mitochondria. The widths of these structures (highlighted with the yellow line) were measured to be ca. 330 and 370 nm respectively, by fitting the intensity profile against Gaussian functions (Figure 4d). These values were in good agreement with literature reports36,37 of mitochondria. Figure 4c was enlarged boxed area in Figure 4b. The major fluorophore release sites (on the cross-section) down to 40 nm apart were readily identified (Figure 4g). The intracellular distribution of NOD550 was studied via colocalization analysis under confocal microscopy (Figure 5). NOD550 was inappropriate for direct distribution analysis, owing to its non-fluorescent nature. However, the product of NOD550 photolysis, dye 1, was bright and further evaluated in co-localization analysis. Mitotracker Deep Red was selected as a standard mitochondrial probe for its low overlapping spectra with NOD550. Before imaging, the cells were exposed to UV illumination to induce in situ generation of fluorophore 1 from NOD550 photolysis. The staining from two dyes exhibits a great similarity and the merged images show yellow marked overlapping. (Figure 5c,e). A Pearson's coefficient
of 0.81 suggests that photoproduct of NOD550 specificity stains in cell mitochondria.
Figure 5. Colocalization analysis of NOD550 with Mitotracker Deep Red. (a) The green-channel image of fluorophore 1 from photolysis of NOD550. (b) The red-channel image of Mitotracker Deep Red. (c) Merged image of the greenchannel and red-channel. (d) Bright-field image. (e) The intensity profile of the crossed section in image c. Scale bars: 10 μm. the green and red channels.
The PALM image (Figure 4) and the co-localization study (Figure 5) have unambiguously indicated that the fluorescent product (1) localized in mitochondria. Where does NOD550 localize prior to photo-activation? To validate the mitochondrial specificity of NOD550, two-color localization imaging was performed with NOD550 and Mitotracker Deep Red (Figure 6). The location and morphology of the mitochondria were delineated by the wide field confocal microscopy in Figure 6b. Then, the sample
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
was imaged with localization microscopy and the singlemolecule tracks of each activated dye were followed (Figure 6d). Clearly, the entire molecular tracks from activetion to bleaching of essentially all activated dyes fall in the area of mitochondria highlighted by Mitotracker Deep Red. This strongly suggests that NOD550 also localizes to mitochondria prior to photoactivation.
namics of mitochondria is not clearly defined in this study and warrants further investigation.
Figure 7. Dynamic mitochondrial morphology revealed by PALM imaging of the mitochondria enriched area in HeLa cells incubated with NOD550. Green and blue arrow labeled the fission events. Red and orange arrow labeled the fusion events. Scale bar: 800 nm.
Figure 6. Two color localization imaging reveals the mitochondrial localization of NOD550. (a) PALM imaging of NOD550 inside live HeLa cell. (b) Wide field fluorescent image of Mitotracker Deep Red indicates the mitochondrial area of the same position. (c) Overlay of PALM and the Confocal images shows that the majority of NO releasing sites match the morphology of mitochondria. (d) Overlay of single molecule tracks (n>3 were shown for clarity) and Confocal image. Scale bar: 1.5 μm.
Super-resolution monitoring of the morphological dynamics of mitochondria was then studied. PALM images of a mitochondria-rich area were acquired with a temporal resolution of 10 s over a course of 60 s (Figure 7). Evidently, mitochondria had exhibited significant morphological changes, including dislocations (blue arrow), fusion (red and orange arrow) and fission (green arrow) during this period. For example, the red arrows highlighted a fusion event of two mitochondria ca. 1.6 µm apart from each other. This fusion was completed within the initial 20 s. Compared to the existing chemical probes for super-resolution monitoring of the mitochondria dynamics, NOD550 is attractive from the following two aspects. First, PALM imaging with NOD550 can be performed in normal culture medium without an oxygen scavenging system or other reducing agents. This allows study of cells in their physiological state. Second, NOD550 makes it possible to monitor the subsequent changes of mitochondria morphology at relatively high temporal resolution upon releasing nitric oxide, which is a signaling molecule known to play a regulatory role in mitochondria dynamics. Therefore, NOD550 represents a valuable molecular tool in NO biology, allowing localization of NO release and morphological changes to mitochondria to be simultaneously monitoring with sub-diffraction resolution. The potential effects of the release NO on the observed dy-
In summary, we have developed a novel phototriggered NO donor (NOD550). Upon photo-activation by UV light, NOD550 decomposes to concomitantly release two equivalence of NO and a rhodamine dye (1), the bright fluorescence of which could be harnessed to study the localization, dose and kinetics of NO release with microscopic methods. We have noted that the absorption bands of NOD550 and 1 are well resolved and therefore selective excitation of 1 without erroneous activation of NOD550 could be achieved. Also, super resolution imaging of cellular NOD550 preferentially exhibits mitochondrial specificity and therefore motion blurring could be minimized. These two aforementioned premises has enabled us to map the localization of NOD550 activation, and hence NO release, with photo-activation localization microscopy (PALM) with spatial resolution of ca. 19 nm and a temporal resolution of ca. 10 s. Mitochondria morphology and even dynamics are readily revealed. We anticipate NOD550 to be a unique and valuable molecular tool for chemical biology. EXPERIMENTAL SECTION Cell culture. HeLa (helacyton gartleri) cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The cells were all maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone). The cells were cultured in a humidified atmosphere of 5% CO2/95% air at 37 oC (CO2 incubator, Thermo Scientific) and grown on 25 mm cover slips (Fisherbrand, 12-545-102) for 1–2 days to reach 70–90% confluency before use. Colocalization imaging. HeLa cells were incubated with 2 μM NOD550 and 0.5 μM Mitotracker Deep Red sequentially and washed with PBS for three times to remove excess dyes. Confocal images were recorded on an Olympus FV1000 confocal microscope. Briefly, two lasers (488 nm for NOD550 and 635 nm for Mitotracker Deep
ACS Paragon Plus Environment
Page 4 of 7
Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry Red) were sequentially applied for imaging to avoid emissive overlap between two dyes. Lasers were focused at the back focal plane of an UPLSAPO 100x oil objective. The fluorescence emission was filtered with a DM 405/488/559/635 and further separate to two channels with SDM 560. The wavelength selection performed by a galvanometer diffraction grating is used as following: 500560 nm for NOD550 and 645-715 nm for Mitotracker Deep Red. The colocalization data was analyzed with the manufacturer's software. Single molecule localization microscope. Imaging were performed on an Olympus IX71 inverted microscope. Two continuous lasers (200 W, Coherent, Sapphire 532200 and 150W, Coherent, OBIS 640-150) were automatically controlled by an acousto-optic tunable filter (AOTF). This laser was further transmitted through an optical fiber, adjusted by motorized-TIRFM illuminator and focused on the back focal plane of an Olympus UAPON 100xo TIRF objective (NA 1.49). Another 375 nm laser (50 mW, MLL-III-375L, Changchun new industries optoelectronics Tech.Co) was controlled by mechanical shutter(Uniblitz) and expanded before injected into the TIRF microscope. The emission from samples was filtered through a Semrock Di01-R405/488/532/635 filter and recorded on an Electron Multiplying CCD (Andor, 897U). Activation analysis. HeLa cells were stained with 5 nM NOD550 for 5 min and then washed with PBS for three times. The images were recorded at a frame rate of 20 Hz. During imaging, the 532 nm laser (30 W/cm2) was continually illuminated the sample for both detecting and bleaching the fluorescent molecules. The other 375 nm laser (10 W/cm2) was manually turned on every 20 seconds to trigger the NO release of NOR550. The point spread functions (PSF) on raw frames were further analyzed with ThunderStorm38. PALM imaging. HeLa cells were stained with 500 nM NOD550 for 5 min and then washed with PBS for three times. The final imaging medium was RPMI 1640 without phenol red (Macgene) supplemented with 10% FBS (Hyclone). Firstly, a wide-field image was recorded with < 1W/cm2 low laser light. During imaging, the sample was continually activated with a ~0.7 W/cm2 375 nm laser and excited with a ~2 kW/cm2 532 nm laser. 1500 images were acquired at 150 Hz. Two color localization imaging. The microscope was the same one as described earlier in single molecule localization microscope section except an implementation of a dual channel emission field splitter (OptoSplit II, Cairn Optics) before detected via the camera. The fluorescence from dye 1 and Mitotracker Deep Red was separated by a dichroic mirror (FF640-FDi01, Semrock) and two emission filter (FF01-585/40 and FF01-697/58, Semrock) equipped in the splitter into short and long wavelength emission. HeLa cells were simultaneously stained with 1 uM NOD550 and 50 nM Mitotracker Deep Red for 5 min. Then the cells were washed with PBS for three times and incubated with DMEM without phenol red (Macgene)
supplemented with 10% FBS (Hyclone). The wide-field image of Mitotracker Deep Red and the PALM imaging of NOD550 were performed simultaneously with two lasers on (~2 kW/cm2 532 nm laser and < 1W/cm2 low 647 nm light laser). The exposure time for each raw super resolution frame (in the short wavelength channel) and the raw wide-field image (in the long wavelength channel) was 0.008s. 1000 raw frame was obtained and further analyzed. The PALM imaging analysis is shown in the single molecule analysis section while first 10 raw wide-field image frames (the remaining frames were excluded for photobleaching) was summed to obtain the final wide-field image of mitochondria. Single molecule analysis. Single molecule and super resolution imaging analysis was performed in ThunderStorm. Briefly, the raw frames were filtered with difference-of-Gaussians filter to select the PSF candidates (For activation analysis, a B-Spline wavelet filter was used for evaluating the point spread functions (PSF) candidates). Then those PSFs were fitted with an integrated form of symmetric 2D Gaussian function (Fitting radius: 3.0 pixel) following Maximum likelihood method39-40 to gave the expected the precise location and single molecule intensity. The localization precision was calculated according to the Thompson formula41. Those PSFs with too low photons emitted (< 500), too high photons emitted (> 10000) or worse localization precision (> 50) are eliminated. In some single molecule localization microscopies, a single molecule PSF might exist across several frames before finally bleached. To avoid the excess localizations of a single fluorophore during imaging, the fitting locations in sequential frames within one-pixel size (160 nm) were merged. The new position was given as the mean lateral coordinates of the successive localizations from the same fluorophore. The single molecule tracking was analyzed with U-Tracker42. The photons emitted from single molecule could be converted from single molecule intensity through the equation below: Photons = (Isig-Ibg)*ADU/(QE*EMGAIN) Isig is the single molecule intensity from the fitting result. Ibg is the background intensity. ADU, the CCD sensitivity, and QE, the quantum efficiency of the camera, are read from the camera manufacturer’s performance sheet. EMGAIN is the gain value used in the experiments (300 in this experiment).
ASSOCIATED CONTENT Supporting Information Supporting Information. General methods, experimental, synthesis, characterizations, tabulated photophysical data, additional spectral data, crystal structure of NOD550, chemostability study, cell cytotoxicity, 1H-NMR, 13C-NMR and HRMS spectra. The Supporting Information is available free of charge on the ACS Publications website. Supporting information (PDF) The crystal structure of NOD550 (CIF)
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
AUTHOR INFORMATION Corresponding Author Email:
[email protected] (Yang, Y.); Email:
[email protected] (Xiao, Y.); Email:
[email protected] (Yang, W.); Email:
[email protected] (Qian, X.).
Author Contributions H. He and Z. Ye contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
ACKNOWLEDGMENT The work is financially supported by the National Natural Science Foundation of China (Nos. 21372080, 21236002, 21376038, 21421005, 21572061, 21576040 and 21776037), National Basic Research Program of China (no. 2013CB733702) and the Fundamental Research Funds for the Central Universities (Nos. WY1514053, WY1516017 and DUT17LK43).
REFERENCES [1]. Detmer, S. A.; Chan, D. C. Nat. Rev. Mol. Cell Biol. 2007, 8, 870-879. [2]. Chen, H.; Chan, D. C. Hum. Mol. Genet. 2009, 18, R169R176. [3]. Archer, S. L. N. Engl. J. Med. 2013, 369, 2236-2251. [4]. Ignarro, L. J. Annu. Rev. Pharmacol. Toxicol. 1990, 30, 535560. [5]. Garthwaite, J.; Boulton, C. L. Annu. Rev. Physiol. 1995, 57, 683-706. [6]. Bogdan, C. Nat. Immun. 2001, 2, 907-916. [7]. Nisoli, E.; Carruba, M. O. J. Cell Sci. 2006, 119, 2855-2862. [8]. Barsoum, M. J.; Yuan, H.; Gerencser, A. A.; Liot, G.; Kushnareva, Y.; Gräber, S.; Kovacs, I.; Lee, W. D.; Waggoner, J.; Cui, J.; White, A. D.; Bossy, B.; Martinou, J.-C.; Youle, R. J.; Lipton, S. A.; Ellisman, M. H.; Perkins, G. A.; Bossy-Wetzel, E. EMBO J. 2006, 25, 3900-3911. [9]. Cho, D.-H.; Nakamura, T.; Fang, J.; Cieplak, P.; Godzik, A.; Gu, Z.; Lipton, S. A. Science. 2009, 324, 102-105. [10]. Westermann, B. Sci. Signal. 2009, 2, pe29. [11]. Nisoli, E.; Clementi, E.; Paolucci, C.; Cozzi, V.; Tonello, C.; Sciorati, C.; Bracale, R.; Valerio, A.; Francolini, M.; Moncada, S.; Carruba, M. O. Science 2003, 299, 896-899. [12]. Leary, S. C.; Shoubridge, E. A. Bioessays. 2003, 25, 538-541. [13]. Nakamura, T.; Cieplak P.; Cho, D. H.; Godzik, A.; Lipton, S. A. Mitochondrion. 2010, 10, 573-578. [14]. Reddy, P. H.; Reddy, T. P.; Manczak, M.; Calkins, M. J.; Shirendeb, U.; Mao, P. Brain Res. Rev. 2011, 67, 103-118. [15]. Haun, F.; Nakamura, T.; Shiu, A. D.; Cho, D. H.; Tsunemi, T.; Holland, E. A.; La Spada, A. R.; Lipton, S. A. Antioxid. Redox Signal. 2013, 19, 1173-1184. [16]. Patterson, G.; Davidson, M.; Manley, S.; LippincottSchwartz, J. Annu. Rev. Phys. Chem. 2010, 61, 345-367. [17]. Jakobs, S.; Wurm, C. A. Curr. Opin. Chem. Biol. 2014, 20, 9-15.
[18]. Rust, M. J.; Bates, M.; Zhuang, X. Nat. Methods 2006, 3, 793-795. [19]. Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; LippincottSchwartz, J.; Hess, H. F. Science 2006, 313, 1642-1645. [20]. Klar, T. A.; Jakobs, S.; Dyba, M.; Egner, A.; Hell, S. W. Proc. Natl. Acad. Sci. USA 2000, 97, 8206-8210. [21]. Shim, S.-H.; Xia, C.; Zhong, G.; Babcock, H. P.; Vaughan, J. C.; Huang, B.; Wang, X.; Xu, C.; Bi, G.-Q.; Zhuang, X. Proc. Natl. Acad. Sci. 2012, 209, 13978-13983. [22]. Carlini, L.; Manley, S. ACS Chem. Biol. 2013, 8, 2643-2648. [23]. Lo, C. Y.-W.; Chen, S.; Creed, S. J.; Kang, M.; Zhao, N.; Tang, B. Z.; Elgass, K. D. Sci. Reports 2016, 6, 30855. [24]. Gu, X.; Zhao, E.; Zhao, T.; Kang, M.; Gui, C.; Lam, J. W. Y.; Du, S.; Loy, M. M. T.; Tang, B. Z. Adv. Mater. 2016, 28, 50645071. [25]. Tullett, J. M.; Rees, D. D. Use of NO Donors in Biological Systems. In Nitric Oxide Protocols; Titheradge, M. A., Ed.; Methods in Molecular Biology series 100; Humana Press: Totowa, NJ, 1998; pp 205-214. [26]. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Chem. Rev. 2002, 102, 1091−1134. [27]. Nitric Oxide Donors: For Pharmaceutical and Biological Applications; Wang, P. G., Cai, T. B., Taniguchi, N., Eds.; WileyVCH: New York, 2005. [28]. Suzuki, T.; Nagae, O.; Kato, Y.; Nakagawa, H.; Fukuhara, K.; Miyata, N. J. Am. Chem. Soc. 2005, 127, 11720-11726. [29]. Rose, M. J.; Olmstead, M. M.; Mascharak, P. K. J. Am. Chem. Soc. 2007, 129, 5342–5343. [30]. Kishikawa, K.; Nakagawa, H.; Furata, T.; Fukuhara, K.; Tsumoto, H.; Suzuki, T.; Miyata, N. J. Am. Chem. Soc. 2009, 131, 74888-7489. [31]. Ieda, N.; Hotta, Y.; Miyata, N.; Kimura, K.; Nakagawa, H. J. Am. Chem. Soc., 2014, 136, 7085-7091. [32]. Kitamura, K.; Kawaguchi, M.; Ieda, Miyata, N.; Nakagawa, H. ACS Chem. Biol. 2016, 11, 1271-1278. [33]. Zhang, Z.; Wu, J.; Shang, Z.; Wang, C.; Cheng, J.; Qian, X.; Xiao, Y.; Xu, Z.; Yang, Y. Anal. Chem. 2016, 88, 7274-7280. [34]. Konopka, C. A.; Bednarek, S. Y. Plant J. 2008, 53,186-196. [35]. Tokunaga, M.； Imamoto, N.; Sakata-Sogawa，K. Nat. Methods, 2008, 5, 159-161. [36]. Patterson, G.; Davidson, M.; Manley, S.; LippincottSchwartz, J. Annu. Rev. Phys. Chem. 2010, 61, 345-367. [37]. Jakobs, S.; Wurm, C. A. Curr. Opin. Chem. Biol. 2014, 20, 9-15.
[38]. M. Ovesný, P. Křížek, J. Borkovec, Z. Švindrych, G. M. Hagen, Bioinformatics 2014, 30, 2389-2390. [39]. K. I. Mortensen, L. S. Churchman, J. A. Spudich, H. Flyvbjerg, Nat. Methods 2010, 7, 377-381. [40]. C. S. Smith, N. Joseph, B. Rieger, K. A. Lidke, Nat. Methods 2010, 7, 373-375. [41]. R. E. Thompson, D. R. Larson, W. W. Webb, Biophys. J. 2002, 82, 2775-2783. [42]. K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods, 2008, 5, 695– 702.
ACS Paragon Plus Environment
Page 6 of 7
Page 7 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications.
7 ACS Paragon Plus Environment
