Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.

Fluorescence labeling and imaging have become the most promising techniques for NO sensing because of their selectivity, sensitivity, and spatiotemporal resolution. Well-developed, small-molecule-based fluorescent probes for NO have beenpromoted, including diaminoaromatic fluorescent compounds [10][11][12] and copper-fluorescein complex [6]. These probes respond specifically, rapidly, and directly to NO at low concentrations, as well as allow NO visualization in single cell. However, none of them can be used to monitor the subcellular distribution of NO, which depends on membrane permeability and NO reactivity, as well as the reduction-oxidation states of intracellular compartments. In our previous study, we observed that protein S-nitrosation derived from endogenous NO production mainly exists in the mitochondria and peri-mitochondrial compartment [13].
To further elucidate the complex biological functions of NO, an ideal NO probe should be able to report the local and subcellular changes in NO concentration. HaloTag is a specific and covalent protein-labeling technology that employs alkyl chloride as reactive moiety, which can covalently bind to a modified bacterial haloalkane dehalogenase (HaloTag) [14,15]. Various HaloTag ligands have been developed for targeted protein labeling [15], protein immobilization [16], super-resolution imaging [17], and magnetic resonance imaging [18]. Based on this specific protein-ligand interaction, we synthesized a novel organelle-targetable fluorescent probe, HTDAF-2DA, which could be used for real-time imaging of NO with fine temporal and spatial resolutions in living cells.

Characterization of HTDAF-2 in vitro
HTDAF-2 was stored at -20°C in the dark until analysis. The stock solutions of NO donor and HTDAF-2 were diluted with PBS (pH 7.4). Fluorescence spectra were measured by a Cary Eclipse spectrofluorometer (Varian). For excitation scans, the emission wavelength was set to 525 nm while scanning the excitation spectra at 1 nm increments from 400 nm to 500 nm. For emission scans, the excitation wavelength was set to 480 nm while scanning the emission spectra at 1 nm increments from 500 nm to 600 nm. The fluorescence response of HTDAF-2 to NO donor (or other chemicals) at varied concentrations was measured by a Synergy 2 Multimode Microplate Reader with excitation filter 485 BP 20 nm and emission filter 528 BP 20 nm (BioTek). Each assay was performed with 25 μL of compounds and 50 μL of HTDAF-2 in a 384-well flat-bottom microplate (Greiner). Fluorescence intensity was measured immediately.

Cell culture
HeLa and MCF-7 cells were grown in DMEM with 10% FBS at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Raw 264.7 Macrophage cells were maintained in RPMI-1640 with 10% FBS. Cells were plated in antibiotic-free medium supplemented with 10% FBS 16 h before transfection. Cells were transfected using FuGene HD transfection reagent (Promega) in accordance to the methods of the manufacturer.

Live-cell fluorescence measurement using a microplate reader
HeLa and MCF-7 cells were incubated at 37°C in DMEM containing 5 μM HTDAF-2DA for loading 36 h after transfection. After 15 min, cells were rinsed four times with PBS and twice with DMEM, as well as incubated in DMEM for 1 h. During incubation, the medium was replaced every 20 min. The medium was then replaced with PBS or PBS with NO donor, and the cells were used for detection. Raw 264.7 cells were stimulated for 8 h with LPS (0.5 μg/ml) plus IFN-γ (250 U/ml) with or without L-NAME (2 mM) 24 h after transfection. Cells were labelled with 5 μM HTDAF-2DA for 30 min and rinsed six times with medium, as well as incubated in RPMI 1640 medium for 1 h. Fluorescence excitation at 485 nm was measured by a Synergy 2 Multi-mode Microplate Reader with excitation filter 485 BP 20 nm and emission filter 528 BP 20 nm. Fluorescence values were background-corrected by subtracting the intensity of HeLa cells that did not express HaloTag protein.

Imaging of HaloTag protein labeled with HTDAF-2DA in living cells
HeLa cells were placed on 35 mm glass-bottom culture dishes (NEST Biotechnology Co. Ltd.) in DMEM supplemented with 10% (v/v) FBS, and observed 36 h post-transfection. HaloTag proteins were expressed in different subcellular compartments by tagging with organellespecific signal peptides. Cells were labeled in accordance to our previously described procedure. For HTDAF-2DA co-labeling with nuclear dye DAPI, cells were first labeled by 5 μM HTDAF-2DA as described and then by 75 μM DAPI for 30 min at 37°C. For co-labeling of HTDAF-2DA, DAPI and MitoTracker Red FM, cells were first labeled by 5 μM HTDAF-2DA as described and then by 75 μM DAPI and 300 nM MitoTracker Red FM. Images were acquired using a high-performance fluorescent microscopy system equipped with a Nikon Eclipse Ti-E automatic microscope, a cooled monochrome digital camera head DS-Qi1 Mc-U2, and a highly stable Shutter Lambda XL light source. A Plan Apo 40×0.95 NA objective was used. For imaging, 482 BP 35 nm band-pass excitation filter and 535/40 emission filter altered by a Lambda 10-XL filter wheel (Shutter Instruments) were used. Images were captured in 640 × 480 format, 12 bit depth, and 60 ms exposure for the channel.

Statistical analysis
Data are presented either as a representative example of a single experiment repeated at least in triplicate or as three or more experiments. Data obtained are represented as mean values ± SD. All P values were obtained using unpaired two-tailed Student's t-test. Values of p<0.05 were considered statistically significant ( Ã 0.01 < p < 0.05; ÃÃ 0.001 < p < 0.01; ÃÃÃ p < 0.001).

Results and Discussions
Design and synthesis of HTDAF-2DA We designed and synthesized HTDAF-2DA for the selective detection of NO. HTDAF-2DA was synthesized in three steps (Scheme A in S1 File). The probe was a monoalkylated derivative of DAF-2DA, a small-molecule fluorescent NO probe widely used for detecting and imaging NO in living cells [10]. Similar to DAF-2 DA [34], the diacetate groups of HTDAF-2DA can be hydrolyzed by esterases after they permeate the cells. The produced HTDAF-2 can then be immobilized by the HaloTag protein, which can be targeted into different subcellular compartments [15] and reacted with NO under aerobic conditions to form a triazole product (Fig 1). This product suppresses the photo-induced electron transfer process and activates the fluorescence of the probe. Another compound, HTFAM, was synthesized and used as a control (Scheme B in S1 File). HTFAM has a structure similar to that of HTDAF-2 but does not react with NO.

Characterization of HTDAF-2DA
In vitro studies showed that HTDAF-2DA exhibited weak fluorescence. However, when the diacetate groups were hydrolyzed in the presence of 1 N NaOH, the fluorescence markedly increased (S1A Fig). HTDAF-2 displayed maximum fluorescence excitation and emission at 490 and 510 nm (Fig 2A), which substantially increased upon the addition of NO donor (MAHMA NONOate and DEA NONOate) (Fig 2B and 2C). In the control, the fluorescence of HTFAM did not respond to the NO donor ( Fig 2B). The fluorescence intensity of HTDAF-2 did not respond noticeably to other reactive oxygen species and reactive nitrogen species, such as O 2 − , H 2 O 2 , NO 2− , and NO 3− (Fig 2C). These results indicated that HTDAF-2 was a highly specific fluorescent probe for NO. The fluorescence of HTDAF-2 and its product after reacting with NO was insignificantly sensitive to pH 7.0 to 8.0 (Fig 2D and S1B Fig), which allowed the determination of NO levels in a physiological environment. These data showed that HTDAF-2 was a highly sensitive and selective fluorescent probe for NO, and it could be used for subsequent experiments in living cells.

Subcellular detection of NO using HTDAF-2DA
In HeLa (cervical cancer cell line) cells, we targeted HaloTag to various subcellular compartments by tagging the protein with or without organelle-specific signal peptides, including the plasma membrane [27][28][29], cytosol [20][21][22][23], nucleus [24][25][26], and mitochondria [30][31][32][33]. After exposing the cells to HTDAF-2DA, washing away the unbound dye, and observing by fluorescent microscopy, we found that the HTDAF-2DA-labeled HaloTag protein produced excellent subcellular localization, which colocalized well with the red fluorescent protein mCherry fused with the same signal peptides (Fig 3A-3D). The correct localizations of HTDAF-2DA-labeled HaloTag protein can also be visualized in fluorescent microscopy images of cells co-stained with the blue fluorescent DNA staining dye DAPI or MitoTracker Red FM (S2A-S2D Fig). By contrast, DAF-2DA probe showed a low spatial resolution (S2E Fig). These data demonstrated that HTDAF-2DA was cell-permeable and could be applied to label specific organelles in living cells. We further analyzed the lysate of organelle HTDAF-2DA-stained cells with or without targeted HaloTag overexpression, by using denatured PAGE. A highly fluorescent band was observed only in HaloTag-overexpressed cells (Fig 3E), which indicated that HTDAF-2DA effectively, specifically, and covalently labeled the HaloTag protein in different subcellular compartments. Compared with other affinity-based tags, covalent labeling provides several advantages, including extending the labeling periods without dissociation of the label, imaging in chemically fixed cells, tolerating stringent wash conditions by immobilizing HaloTag on solid supports, and allowing multiplexing with immunocytochemical methods, SDS-PAGE, and western blot analysis [15,35].
NO and NO synthases are ubiquitous in malignant tumors and are known to exert pro-and anti-tumor effects [3,36,37]. To understand NO responses in cancer biology, we analyzed HTDAF-2 fluorescence in different subcellular compartments of HeLa and MCF-7 (breast cancer cell lines). The results showed that exogenous NO addition led to the largest fluorescence changes in the membrane, followed by those in the cytosol, nucleus, and mitochondria ( Fig  3F-3H). These results demonstrated that cell-permeable HTDAF-2DA could be used for in situ imaging of NO in living cells and tracing NO changes in different subcellular organelles. Moreover, the cell membrane was highly enriched with NO species.
It is well known that NO is produced by induced nitric oxide synthase in stimulated macrophages, which was monitored by using the Griess assay or NO fluorescent probes [6,[38][39][40]. However, these methods did not reveal intracellular NO production with spatial resolution.  HTDAF-2DA, targeted to cytosol/nucleus or nucleus alone, readily detects subcellular NO produced in Raw 264.7 murine macrophages prestimulated with LPS and IFN-γ (Fig 4A and 4B). Furthermore, L-NAME, a known inhibitor of nitric oxide synthase, significantly decreased the increase of HTDAF-2DA fluorescence in LPS-and IFN-γ-treated macrophages, in agreement with previous reports [38][39][40]. Similar results were obtained when the production of NO was monitored using the widely used NO probe DAF-2DA (Fig 4C).
Direct determination of the spatiotemporal distribution of NO in situ is considerably significant in the exact function of NO in physiological and pathological conditions. However, convincing methods that provide spatiotemporal information of NO signaling are not yet available. Srikun et al. reported some organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-Tag protein labelling [41]. In this paper, we report a new organelle-targetable NO probe via HaloTag protein labeling. The presented methodology could also be extended to other small-molecule probes with genetically encoded protein scaffolds (e.g., HaloTag, SNAP-tag, and CLIP-tag).
In summary, we have described the design, synthesis, spectroscopic properties, NO responses, and subcellular NO imaging applications of a HaloTag-based fluorescent probe called HTDAF-2DA. This hybrid small-molecule/protein reporter could be used to label various subcellular compartments, including the plasma membrane, cytosol, nucleus, and mitochondria, as well as measure changes in local NO fluxes in living cells by microscopy. Several studies have demonstrated that high levels of NO can trigger cancer cell death via DNA damage, oxidative/nitrosative stress, cytotoxicity, and apoptosis [36,42]. Various approaches for cancer treatment have been investigated, including NO-releasing drugs and NO as chemotherapy and radiotherapy sensitizers. Our results show that exogenous NO mainly accumulated in cancer cell membrane, which suggests that NO-triggered membrane damage may be the vanguard event of NO-induced cell death. The capability to image the spatial distribution of subcellular NO real time could be useful to better understand cellular signaling involving NO, including guanylyl cyclase activation, vessel homeostasis, neurotransmission, ischemia, inflammation, and neurodegeneration [43,44]. HTDAF-2DA may also serve as a valuable tool for highthroughput screening of NO-donating drugs in cancer therapy. Further studies on expanding  the color palette of targetable NO probes, optimizing the sensitivities and dynamic range of NO probes, and creating targetable sensors with ratiometric read-out are currently in progress.