Skip to main content

Intracellular pH Measurements In Vivo Using Green Fluorescent Protein Variants

  • Protocol
C. elegans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 351))

Abstract

Whether by patch-clamp techniques or the use of fluorescent vital dyes, measurements of transepithelial ion flux in mammals are limited by cell accessibility. Furthermore, redundant functions and complex regulatory mechanisms can mask loss-of-function phenotypes through compensatory mechanisms. In this chapter, we present a technique whereby the optically transparent nematode Caenorhabditis elegans, engineered to express a fluorescent pH indicator protein, can be used to study how intracellular pH (pHi) fluctuates in response to environmental and/or experimental challenge. By using a live whole animal model, systemic, and even behavioral relationships to individual cellular pHi can be inferred. In combination with dye loading of excised or cultured cells, this technique also provides a powerful means of contrasting these relationships to biophysical measurements of ion flux.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Verkman, A. S., Sellers, M. C., Chao, A. C., Leung, T., and Ketcham, R. (1989) Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications. Anal. Biochem. 178, 355–361.

    Article  PubMed  CAS  Google Scholar 

  2. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450.

    PubMed  CAS  Google Scholar 

  3. Rink, T. J., Tsien, R. Y., and Pozzan, T. (1982) Cytoplasmic pH and free Mg2+ in lymphocytes. J. Cell Biol. 95, 189–196.

    Article  PubMed  CAS  Google Scholar 

  4. Orlowski, J. and Grinstein, S. (2004) Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch. 447, 549–565.

    Article  PubMed  CAS  Google Scholar 

  5. Bokman, S. H. and Ward, W. W. (1981) Renaturation of Aequorea green-fluorescent protein. Biochem. Biophys. Res. Commun. 101, 1372–1380.

    Article  PubMed  CAS  Google Scholar 

  6. Ward, W. W. and Bokman, S. H. (1982) Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21, 4535–4540.

    Article  PubMed  CAS  Google Scholar 

  7. Kneen, M., Farinas, J., Li, Y., and Verkman, A. S. (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys. J. 74, 1591–1599.

    Article  PubMed  CAS  Google Scholar 

  8. Llopis, J., McCaffery, J. M., Miyawaki, A., Farquhar, M. G., and Tsien, R. Y. (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA 95, 6803–6808.

    Article  PubMed  CAS  Google Scholar 

  9. Elsliger, M. A., Wachter, R. M., Hanson, G. T., Kallio, K., and Remington, S. J. (1999) Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry 38, 5296–5301.

    Article  PubMed  CAS  Google Scholar 

  10. Takahashi, A., Zhang, Y., Centonze, E., and Herman, B. (2001) Measurement of mitochondrial pH in situ. BioTechniques 30, 804–808.

    PubMed  CAS  Google Scholar 

  11. Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., and Reed, J. C. (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat. Cell. Biol. 2, 318–325.

    Article  PubMed  CAS  Google Scholar 

  12. Miesenbock, G., De Angelis, D. A., and Rothman, J. E. (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195.

    Article  PubMed  CAS  Google Scholar 

  13. Hanson, G. T., McAnaney, T. B., Park, E. S., et al. (2002) Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 41, 15,477–15,488.

    Article  PubMed  CAS  Google Scholar 

  14. Sankaranarayanan, S. and Ryan, T. A. (2001) Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat. Neuroscience. 4, 129–136.

    Article  CAS  Google Scholar 

  15. Ohara-Imaizumi, M., Nakamichi, Y., Tanaka, T., Katsuta, H., Ishida, H., and Nagamatsu, S. (2002) Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy. Biochem. J. 363, 73–80.

    Article  PubMed  CAS  Google Scholar 

  16. Poskanzer, K. E., Marek, K. W., Sweeney, S. T., and Davis, G. W. (2003) Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature 426, 559–563.

    Article  PubMed  CAS  Google Scholar 

  17. Mitchell, S. J. and Ryan, T. A. (2004) Syntaxin-1A is excluded from recycling synaptic vesicles at nerve terminals. J. Neurosci. 24, 4884–4888.

    Article  PubMed  CAS  Google Scholar 

  18. Ashby, M. C., De La Rue, S. A., Ralph, G. S., Uney, J., Collingridge, G. L., and Henley, J. M. (2004) Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. J. Neurosci. 24, 5172–5176.

    Article  PubMed  CAS  Google Scholar 

  19. Nehrke, K. (2003) A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span. J. Biol. Chem. 278, 44,657–44,666.

    Article  PubMed  CAS  Google Scholar 

  20. Vijayvergiya, C., De Angelis, D., Walther, M., et al. (2004) High-level expression of rabbit 15-lipoxygenase induces collapse of the mitochondrial pH gradient in cell culture. Biochemistry 43, 15,296–15,302.

    Article  PubMed  CAS  Google Scholar 

  21. Estevez, A. Y., Roberts, R. K., and Strange, K. (2003) Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells. J. Gen. Physiol. 122, 207–223.

    Article  PubMed  CAS  Google Scholar 

  22. Christensen, M., Estevez, A., Yin, X., et al. (2002) A primary culture system for functional analysis of C. elegans neurons and muscle cells. Neuron 33, 503–514.

    Article  PubMed  CAS  Google Scholar 

  23. Bamber, B. A., Richmond, J. E., Otto, J. F., and Jorgensen, E. M. (2005) The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction. Br. J. Pharmacol. 144, 502–509.

    Article  PubMed  CAS  Google Scholar 

  24. Machaca, K., DeFelice, L. J., and L’Hernault, S. W. (1996) A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation. Dev. Biol. 176, 1–16.

    Article  PubMed  CAS  Google Scholar 

  25. Goodman, M. B., Hall, D. H., Avery, L., and Lockery, S. R. (1998) Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron 20, 763–772.

    Article  PubMed  CAS  Google Scholar 

  26. Richmond, J. E. and Jorgensen, E. M. (1999) One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 2, 791–797.

    Article  PubMed  CAS  Google Scholar 

  27. Rutledge, E., Bianchi, L., Christensen, M., et al. (2001) CLH-3, a ClC-2 anion channel ortholog activated during meiotic maturation in C. elegans oocytes. Curr. Biol. 11, 161–170.

    Article  PubMed  CAS  Google Scholar 

  28. Jospin, M., Mariol, M. C., Sâegalat, L., and Allard, B. (2002) Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans. J. Physiol. 544, 373–384.

    Article  PubMed  CAS  Google Scholar 

  29. Jospin, M. and Allard, B. (2004) An amiloride-sensitive H+-gated Na+ channel in Caenorhabditis elegans body wall muscle cells. J. Physiol. 559, 715–720.

    PubMed  CAS  Google Scholar 

  30. Kerr, R., Lev-Ram, V., Baird, G., Vincent, P., Tsien, R. Y., and Schafer, W. R. (2000) Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594.

    Article  PubMed  CAS  Google Scholar 

  31. Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210–2218.

    Article  PubMed  CAS  Google Scholar 

  32. Bozza, T., McGann, J. P., Mombaerts, P., and Wachowiak, M. (2004) In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron 42, 9–21.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Nehrke, K. (2006). Intracellular pH Measurements In Vivo Using Green Fluorescent Protein Variants. In: Strange, K. (eds) C. elegans. Methods in Molecular Biology, vol 351. Humana Press. https://doi.org/10.1385/1-59745-151-7:223

Download citation

  • DOI: https://doi.org/10.1385/1-59745-151-7:223

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-597-2

  • Online ISBN: 978-1-59745-151-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics