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Real-time intravital imaging of pH variation associated with osteoclast activity

Abstract

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

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Figure 1
Figure 2: Chemical structures and spectroscopic data of pHocas-1–3 and pHocas-AL.
Figure 3: Evaluation of the availability of pHocas-3 for TPEM imaging of osteoclast activity in living mice.
Figure 4: Quantitative analysis of osteoclast activity in vivo based on colocalization analysis of pHocas-3 and tdTomato.
Figure 5: Two-photon time-lapse imaging of bone tissues, acquired at 5-min intervals for 8 h after injection of pHocas-3.
Figure 6: Relationship between the formation of an acidic compartment and cell motility.

References

  1. Giampieri, S. et al. Localized and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility. Nat. Cell Biol. 11, 1287–1296 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. van Panhuys, N., Klauschen, F. & Germain, R.N. T-cell-receptor-dependent signal intensity dominantly controls CD4+ T cell polarization in vivo. Immunity 41, 63–74 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Ishii, M. et al. Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature 458, 524–528 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang, W. et al. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res. 62, 6278–6288 (2002).

    CAS  PubMed  Google Scholar 

  5. Overstreet, M.G. et al. Inflammation-induced interstitial migration of effector CD4 T cells is dependent on integrin αV. Nat. Immunol. 14, 949–958 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Bousso, P., Bhakta, N.R., Lewis, R.S. & Robey, E. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).

    CAS  PubMed  Google Scholar 

  7. Miller, M.J., Wei, S.H., Parker, I. & Cahalan, M.D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    CAS  PubMed  Google Scholar 

  8. Stoll, S., Delon, J., Brotz, T.M. & Germain, R.N. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).

    PubMed  Google Scholar 

  9. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    CAS  PubMed  Google Scholar 

  10. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Wu, J. et al. A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging. Nat. Commun. 5, 5262 (2014).

    CAS  PubMed  Google Scholar 

  12. Liu, D. et al. T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction. Nature 517, 214–218 (2015).

    CAS  PubMed  Google Scholar 

  13. Nobis, M. et al. Intravital FLIM-FRET imaging reveals dasatinib-induced spatial control of src in pancreatic cancer. Cancer Res. 73, 4674–4686 (2013).

    CAS  PubMed  Google Scholar 

  14. Johnsson, A.K. et al. The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Rep. 6, 1153–1164 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mizuno, R. et al. In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J. Exp. Med. 211, 1123–1136 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Breart, B., Lemaître, F., Celli, S. & Bousso, P. Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J. Clin. Invest. 118, 1390–1397 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Chittajallu, D.R. et al. In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy. Nat. Methods 12, 577–585 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lavis, L.D. & Raines, R.T. Bright ideas for chemical biology. ACS Chem. Biol. 3, 142–155 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Qi, H., Egen, J.G., Huang, A.Y.C. & Germain, R.N. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312, 1672–1676 (2006).

    CAS  PubMed  Google Scholar 

  21. Hilderbrand, S.A. & Weissleder, R. Near-infrared fluorescence: application to in vivo molecular imaging. Curr. Opin. Chem. Biol. 14, 71–79 (2010).

    CAS  PubMed  Google Scholar 

  22. Boyle, W.J., Simonet, W.S. & Lacey, D.L. Osteoclast differentiation and activation. Nature 423, 337–342 (2003).

    CAS  PubMed  Google Scholar 

  23. Kowada, T. et al. In vivo fluorescence imaging of bone-resorbing osteoclasts. J. Am. Chem. Soc. 133, 17772–17776 (2011).

    CAS  PubMed  Google Scholar 

  24. Kikuta, J. et al. Dynamic visualization of RANKL and Th17-mediated osteoclast function. J. Clin. Invest. 123, 866–873 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kozloff, K.M., Volakis, L.I., Marini, J.C. & Caird, M.S. Near-infrared fluorescent probe traces bisphosphonate delivery and retention in vivo. J. Bone Miner. Res. 25, 1748–1758 (2010).

    CAS  PubMed  Google Scholar 

  26. Mula, S. et al. Design and development of a new pyrromethene dye with improved photostability and lasing efficiency: theoretical rationalization of photophysical and photochemical properties. J. Org. Chem. 73, 2146–2154 (2008).

    CAS  PubMed  Google Scholar 

  27. Jones, G. II., Kumar, S., Klueva, S. & Pacheco, D. Photoinduced electron transfer for pyrromethene dyes. J. Phys. Chem. A 107, 8429–8434 (2003).

    CAS  Google Scholar 

  28. Silverton, S. Osteoclast radicals. J. Cell. Biochem. 56, 367–373 (1994).

    CAS  PubMed  Google Scholar 

  29. Halleen, J.M. et al. Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase. J. Biol. Chem. 274, 22907–22910 (1999).

    CAS  PubMed  Google Scholar 

  30. Komatsu, T. et al. Rational design of boron dipyrromethene (BODIPY)-based photobleaching-resistant fluorophores applicable to a protein dynamics study. Chem. Commun. (Camb.) 47, 10055–10057 (2011).

    CAS  Google Scholar 

  31. Sunahara, H., Urano, Y., Kojima, H. & Nagano, T. Design and synthesis of a library of BODIPY-based environmental polarity sensors utilizing photoinduced electron-transfer-controlled fluorescence ON/OFF switching. J. Am. Chem. Soc. 129, 5597–5604 (2007).

    CAS  PubMed  Google Scholar 

  32. Gabe, Y., Urano, Y., Kikuchi, K., Kojima, H. & Nagano, T. Highly sensitive fluorescence probes for nitric oxide based on boron dipyrromethene chromophore-rational design of potentially useful bioimaging fluorescence probe. J. Am. Chem. Soc. 126, 3357–3367 (2004).

    CAS  PubMed  Google Scholar 

  33. Ducros, M. et al. Spectral unmixing: analysis of performance in the olfactory bulb in vivo. PLoS One 4, e4418 (2009).

    PubMed  PubMed Central  Google Scholar 

  34. Salo, J., Lehenkari, P., Mulari, M., Metsikkö, K. & Väänänen, H.K. Removal of osteoclast bone resorption products by transcytosis. Science 276, 270–273 (1997).

    CAS  PubMed  Google Scholar 

  35. Takahashi, N. et al. Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122, 1373–1382 (1988).

    CAS  PubMed  Google Scholar 

  36. Lacey, D.L. et al. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab. Nat. Rev. Drug Discov. 11, 401–419 (2012).

    CAS  PubMed  Google Scholar 

  37. Karsdal, M.A. et al. Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption. Am. J. Pathol. 166, 467–476 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Minkin, C. Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif. Tissue Int. 34, 285–290 (1982).

    CAS  PubMed  Google Scholar 

  39. Tamura, T. et al. New resorption assay with mouse osteoclast-like multinucleated cells formed in vitro. J. Bone Miner. Res. 8, 953–960 (1993).

    CAS  PubMed  Google Scholar 

  40. Entenberg, D. et al. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat. Protoc. 6, 1500–1520 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kim, H.J., Heo, C.H. & Kim, H.M. Benzimidazole-based ratiometric two-photon fluorescent probes for acidic pH in live cells and tissues. J. Am. Chem. Soc. 135, 17969–17977 (2013).

    CAS  PubMed  Google Scholar 

  42. Miao, F. et al. Fluorescent imaging of acidic compartments in living cells with a high selective novel one-photon ratiometric and two-photon acidic pH probe. Biosens. Bioelectron. 50, 42–49 (2013).

    CAS  PubMed  Google Scholar 

  43. Yao, S., Schafer-Hales, K.J. & Belfield, K.D. A new water-soluble near-neutral ratiometric fluorescent pH indicator. Org. Lett. 9, 5645–5648 (2007).

    CAS  PubMed  Google Scholar 

  44. Urano, Y. et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat. Med. 15, 104–109 (2009).

    CAS  PubMed  Google Scholar 

  45. Loudet, A. & Burgess, K. BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 107, 4891–4932 (2007).

    CAS  PubMed  Google Scholar 

  46. Kowada, T., Maeda, H. & Kikuchi, K. BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chem. Soc. Rev. 44, 4953–4972 (2015).

    CAS  PubMed  Google Scholar 

  47. Li, L. et al. A sensitive two-photon probe to selectively detect monoamine oxidase B activity in Parkinson's disease models. Nat. Commun. 5, 3276 (2014).

    PubMed  Google Scholar 

  48. Kavarnos, G.J. Fundamentals of Photoinduced Electron Transfer (VCH, New York, 1993).

  49. Frisch, M.J. et al. Gaussian 09, Revision D.02 (Gaussian, Inc., Wallingford, Connecticut, USA, 2013).

  50. Hilal, S.H., Karickhoff, S.W. & Carreira, L.A. A rigorous test for SPARC's chemical reactivity models: estimation of more than 4300 ionization pKas. Quant. Struct. Act. Relat. 14, 348–355 (1995).

    CAS  Google Scholar 

  51. Plumeré, N., Henig, J. & Campbell, W.H. Enzyme-catalyzed O2 removal system for electrochemical analysis under ambient air: application in an amperometric nitrate biosensor. Anal. Chem. 84, 2141–2146 (2012).

    PubMed  Google Scholar 

  52. Aubry, J.M., Cazin, B. & Duprat, F. Chemical sources of singlet oxygen. 3. Peroxidation of water-soluble singlet oxygen carriers with the hydrogen peroxide-molybdate system. J. Org. Chem. 54, 726–728 (1989).

    CAS  Google Scholar 

  53. Auchère, F., Bertho, G., Artaud, I., Girault, J.P. & Capeillère-Blandin, C. Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system. Eur. J. Biochem. 268, 2889–2895 (2001).

    PubMed  Google Scholar 

  54. Gloire, G., Legrand-Poels, S. & Piette, J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem. Pharmacol. 72, 1493–1505 (2006).

    CAS  PubMed  Google Scholar 

  55. Forman, H.J. & Torres, M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med. 166, S4–S8 (2002).

    PubMed  Google Scholar 

  56. Ishii, M., Kikuta, J., Shimazu, Y., Meier-Schellersheim, M. & Germain, R.N. Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo. J. Exp. Med. 207, 2793–2798 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (grant nos. 25220207, 26102529, and 15K12754 to K.K., 14J00794 to H.M.). K.K. and M.I. thank the Naito Foundation for financial support. K.K. and S.M. thank the Asahi Glass Foundation for financial support. K.K. and M.I. also thank the Uehara Memorial Foundation for financial support.

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Authors and Affiliations

Authors

Contributions

H.M., T.K., S.M., and K.K. contributed to the development of chemical probes, and M.F., M.S., J.K., and M.I. conducted imaging experiments in vivo. H.M. and T.K. synthesized and characterized chemical probes. M.F., M.S., and J.K. performed in vivo studies. H.M. and T.K. co-wrote the initial draft. J.K., M.F., S.M., M.I., and K.K. revised the final draft.

Corresponding authors

Correspondence to Masaru Ishii or Kazuya Kikuchi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–10. (PDF 1095 kb)

Supplementary Note

Synthetic Procedures. (PDF 563 kb)

Real-time fluorescence imaging of osteoclasts in living mice with pHocas-AL.

Long-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-AL was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 5 min for 8 h and processed using spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 7989 kb)

Real-time fluorescence imaging of osteoclasts in living mice with pHocas-3.

Long-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-3 was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 5 min for 8 h and processed using spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 7242 kb)

In vivo imaging of osteoclast activation for short intervals.

Short-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-3 was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 1 min for 130 min without spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 6999 kb)

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Maeda, H., Kowada, T., Kikuta, J. et al. Real-time intravital imaging of pH variation associated with osteoclast activity. Nat Chem Biol 12, 579–585 (2016). https://doi.org/10.1038/nchembio.2096

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