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

Wang J, Zhao Y, Wang C, Zhu Q, Du Z, Hu A, Yang Y - PLoS ONE (2015)

Bottom Line: NO participates and plays important roles in multiple biological processes.However, spatiotemporal imaging of NO in living cells is challenging.The developed strategy enables precise determination of local NO function.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, CAS Center for Excellence in Brain Science, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, P. R. China.

ABSTRACT
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.

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Fluorescence detection of NO in subcellular organelles of HeLa and MCF-7 cells.Targeted localization of HTDAF-2 by conjugation to HaloTag proteins in living HeLa cells. Images present HeLa cells expressing HaloTag in the cytosol/nucleus (A), nucleus (B), membrane (C), and mitochondria (D), with the red fluorescent protein mCherry fused with the same signal peptides. Scale bar = 10 μm. (E) Direct in-gel fluorescence of control (1), nucleus-HaloTag (2), plasma membrane-HaloTag (3), cytosol-HaloTag (4), and mitochondria-HaloTag (5) in HeLa cells labeled with 5 μM HTDAF-2DA. (F) The fluorescence responses of 5 μM HTDAF-2DA targeted in the plasma membrane to various concentrations of NO donor (DEA NONOate) in HeLa cells. (G and H) Kinetics of fluorescence response of 5 μM HTDAF-2DA in different subcellular compartments of HeLa (G) and MCF-7 (H) cells upon the addition of NO donor (DEA NONOate). Error bars represent SD.
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pone.0123986.g003: Fluorescence detection of NO in subcellular organelles of HeLa and MCF-7 cells.Targeted localization of HTDAF-2 by conjugation to HaloTag proteins in living HeLa cells. Images present HeLa cells expressing HaloTag in the cytosol/nucleus (A), nucleus (B), membrane (C), and mitochondria (D), with the red fluorescent protein mCherry fused with the same signal peptides. Scale bar = 10 μm. (E) Direct in-gel fluorescence of control (1), nucleus-HaloTag (2), plasma membrane-HaloTag (3), cytosol-HaloTag (4), and mitochondria-HaloTag (5) in HeLa cells labeled with 5 μM HTDAF-2DA. (F) The fluorescence responses of 5 μM HTDAF-2DA targeted in the plasma membrane to various concentrations of NO donor (DEA NONOate) in HeLa cells. (G and H) Kinetics of fluorescence response of 5 μM HTDAF-2DA in different subcellular compartments of HeLa (G) and MCF-7 (H) cells upon the addition of NO donor (DEA NONOate). Error bars represent SD.

Mentions: 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 [27–29], cytosol [20–23], nucleus [24–26], and mitochondria [30–33]. 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].


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

Wang J, Zhao Y, Wang C, Zhu Q, Du Z, Hu A, Yang Y - PLoS ONE (2015)

Fluorescence detection of NO in subcellular organelles of HeLa and MCF-7 cells.Targeted localization of HTDAF-2 by conjugation to HaloTag proteins in living HeLa cells. Images present HeLa cells expressing HaloTag in the cytosol/nucleus (A), nucleus (B), membrane (C), and mitochondria (D), with the red fluorescent protein mCherry fused with the same signal peptides. Scale bar = 10 μm. (E) Direct in-gel fluorescence of control (1), nucleus-HaloTag (2), plasma membrane-HaloTag (3), cytosol-HaloTag (4), and mitochondria-HaloTag (5) in HeLa cells labeled with 5 μM HTDAF-2DA. (F) The fluorescence responses of 5 μM HTDAF-2DA targeted in the plasma membrane to various concentrations of NO donor (DEA NONOate) in HeLa cells. (G and H) Kinetics of fluorescence response of 5 μM HTDAF-2DA in different subcellular compartments of HeLa (G) and MCF-7 (H) cells upon the addition of NO donor (DEA NONOate). Error bars represent SD.
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Related In: Results  -  Collection

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pone.0123986.g003: Fluorescence detection of NO in subcellular organelles of HeLa and MCF-7 cells.Targeted localization of HTDAF-2 by conjugation to HaloTag proteins in living HeLa cells. Images present HeLa cells expressing HaloTag in the cytosol/nucleus (A), nucleus (B), membrane (C), and mitochondria (D), with the red fluorescent protein mCherry fused with the same signal peptides. Scale bar = 10 μm. (E) Direct in-gel fluorescence of control (1), nucleus-HaloTag (2), plasma membrane-HaloTag (3), cytosol-HaloTag (4), and mitochondria-HaloTag (5) in HeLa cells labeled with 5 μM HTDAF-2DA. (F) The fluorescence responses of 5 μM HTDAF-2DA targeted in the plasma membrane to various concentrations of NO donor (DEA NONOate) in HeLa cells. (G and H) Kinetics of fluorescence response of 5 μM HTDAF-2DA in different subcellular compartments of HeLa (G) and MCF-7 (H) cells upon the addition of NO donor (DEA NONOate). Error bars represent SD.
Mentions: 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 [27–29], cytosol [20–23], nucleus [24–26], and mitochondria [30–33]. 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].

Bottom Line: NO participates and plays important roles in multiple biological processes.However, spatiotemporal imaging of NO in living cells is challenging.The developed strategy enables precise determination of local NO function.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, CAS Center for Excellence in Brain Science, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, P. R. China.

ABSTRACT
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.

Show MeSH