SpringerLink
Log in

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy

  • Protocol
  • First Online:
Dictyostelium discoideum

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2814))

  • 67 Accesses

Abstract

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
EUR 44.95
Price includes VAT (Germany)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 179.99
Price includes VAT (Germany)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
EUR 235.39
Price includes VAT (Germany)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Srinivasan K, Wright GA, Hames N, Housman M, Roberts A, Aufderheide KJ, Janetopoulos C (2013) Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses. J Cell Sci 126:221–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kortholt A, King JS, Keizer-Gunnink I, Harwood AJ, Van Haastert PJ (2007) Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis. Mol Biol Cell 18:4772–4779

    Article  CAS  PubMed  Google Scholar 

  3. Camps M, Carozzi A, Schnabel P, Scheer A, Parker PJ, Gierschik P (1992) Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature 360:684–686

    Article  CAS  PubMed  Google Scholar 

  4. Jiang H, Wu D, Simon MI (1994) Activation of phospholipase C beta 4 by heterotrimeric GTP-binding proteins. J Biol Chem 269:7593–7596

    Article  CAS  PubMed  Google Scholar 

  5. Park D, Jhon DY, Lee CW, Lee KH, Rhee SG (1993) Activation of phospholipase C isozymes by G protein beta gamma subunits. J Biol Chem 268:4573–4576

    Article  CAS  PubMed  Google Scholar 

  6. Li Z, Jiang H, **e W, Zhang Z, Smrcka AV, Wu D (2000) Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. Science 287:1046–1049

    Article  CAS  PubMed  Google Scholar 

  7. Xu X, Gera N, Li H, Yun M, Zhang L, Wang Y, Wang QJ, ** T (2015) GPCR-mediated PLCbetagamma/PKCbeta/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis. Mol Biol Cell 26:874–886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Xu X, Wen X, Bhimani S, Moosa A, Parsons D, Ha H, ** T (2023) G protein-coupled receptor-mediated membrane targeting of PLCγ2 is essential for neutrophil chemotaxis. J Leukoc Biol 114:126–141

    Article  PubMed  Google Scholar 

  9. Tang W, Zhang Y, Xu W, Harden TK, Sondek J, Sun L, Li L, Wu D (2011) A PLCbeta/PI3Kgamma-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis. Dev Cell 21:1038–1050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu L, Gritz D, Parent CA (2014) PKCbetaII acts downstream of Chemoattractant receptors and mtorc2 to regulate cAMP Production and myosin ii activity in neutrophils. Mol Biol Cell 25:1446–1457

    Article  PubMed  PubMed Central  Google Scholar 

  11. Clemens RA, Lowell CA (2015) Store-operated calcium signaling in neutrophils. J Leukoc Biol 98:497–502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wei C, Wang X, Chen M, Ouyang K, Song LS, Cheng H. (2009). Calcium flickers steer cell migration. Nature, 2009. 457:901-905

    Google Scholar 

  13. Evans JH, Falke JJ (2007) Ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages. Proc Natl Acad Sci USA 104:16176–16181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tsai FC, Meyer T (2012) Ca2+ pulses control local cycles of lamellipodia retraction and adhesion along the front of migrating cells. Curr Biol 22:837–842

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xu X, Wen X, Moosa A, Bhimani S, ** T (2021) Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients. Proc Natl Acad Sci USA 118:e2002162118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Traynor D, Milne JL, Insall RH, Kay RR (2000) Ca(2+) signalling is not required for chemotaxis in Dictyostelium. EMBO J 19:4846–48454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Traynor D, Kay RR (2017) polycystin-type transient receptor potential (Trp) channel that is activated by ATP. Biol Open 6:200–209

    CAS  PubMed  Google Scholar 

  18. Xu X, Pots H, Gilsbach BK, Parsons D, Veltman DM, Ramachandra SG, Li H, Kortholt A, ** T (2022) C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis. Front Immunol 13:1075386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xu X, Bhimani S, Pots H, Wen X, Jeon TJ, Kortholt A, ** T (2021) Membrane targeting of C2GAP1 enables Dictyostelium discoideum to sense chemoattractant gradient at a higher concentration range. Front Cell Dev Biol 9:725073

    Article  PubMed  PubMed Central  Google Scholar 

  20. Xu X, Gera N, Li H, Yun M, Zhang L, Wang Y, Wang QJ, ** T (2015) GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis. Mol Biol Cell 26:874–886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tang W, Zhang Y, Xu W, Harden TK, Sondek J, Sun L, Li L, Wu D (2011) A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis. Dev Cell 21:1038–1050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gulyás G, Tóth JT, Tóth DJ, Kurucz I, Hunyady L, Balla T, Várnai P (2015) Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors. PLoS One 10:e0125601

    Article  PubMed  PubMed Central  Google Scholar 

  23. Oura T, Murata K, Morita T, Nezu A, Arisawa M, Shuto S, Tanimura A (2016) Highly sensitive measurement of inositol 1,4,5-trisphosphate by using a new fluorescent ligand and ligand binding domain combination. Chembiochem 17:1509–1512

    Article  CAS  PubMed  Google Scholar 

  24. Tanimura A, Nezu A, Morita T, Turner RJ, Tojyo Y (2004) Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells. J Biol Chem 279:38095–38098

    Article  CAS  PubMed  Google Scholar 

  25. Zigmond SH (1978) Chemotaxis by polymorphonuclear leukocytes. J Cell Biol 77:269–287

    Article  CAS  PubMed  Google Scholar 

  26. Gerisch G, Albrecht R, Heizer C, Hodgkinson S, Maniak M (1995) Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein–coronin fusion protein. Curr Biol 5:1280–1285

    Article  CAS  PubMed  Google Scholar 

  27. Wen X, ** T, Xu X (2016) Imaging G protein-coupled receptor-mediated chemotaxis and its signaling events in neutrophil-like HL60 cells. J Vis Exp 14:54511

    Google Scholar 

  28. Li J, Zhu L, Zhang M, Lin F (2012) Microfluidic device for studying cell migration in single or co-existing chemical gradients and electric fields. Biomicrofluidics 6:24121–2412113

    Article  PubMed  Google Scholar 

  29. Xu X, ** T (2011) Imaging G-protein coupled receptor (GPCR)-mediated signaling events that control chemotaxis of Dictyostelium discoideum. J Vis Exp 55

    Google Scholar 

  30. Xu X, Brzostowski JA, ** T (2006) Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol Biol 346:281–296

    CAS  PubMed  Google Scholar 

  31. Xu X, Yun M, Wen X, Brzostowski J, Quan W, Wang QJ, ** T (2016) Quantitative monitoring spatiotemporal activation of Ras and PKD1 using confocal fluorescent microscopy. Methods Mol Biol 2016(1407):307–323

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank all members of the Chemotaxis Signal Section of the Laboratory of Immunogenetics (CSS/LIG), NIAID, NIH. This research was supported by the Intramural Research Program of the NIH/NIAID. We also thank Gergő Gulyás and Dr. Péter Várnai of Semmelweis University, Budapest, Hungary, for providing IP3 probes for this work.

Author information

Authors and Affiliations

  1. Chemotaxis Signaling Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, MD, USA

    Xuehua Xu, HyunGee Ha & Tian **

  2. Imaging Core Facility, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, MD, USA

    Joseph Brzostowski

Authors
  1. Xuehua Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. HyunGee Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph Brzostowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tian **
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xuehua Xu .

Editor information

Editors and Affiliations

  1. Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD, USA

    Alan R. Kimmel

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Xu, X., Ha, H., Brzostowski, J., **, T. (2024). Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy. In: Kimmel, A.R. (eds) Dictyostelium discoideum. Methods in Molecular Biology, vol 2814. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3894-1_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3894-1_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3893-4

  • Online ISBN: 978-1-0716-3894-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation