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Breast Cancer pp 225-237 | Cite as

Monitoring Phosphatidic Acid Signaling in Breast Cancer Cells Using Genetically Encoded Biosensors

  • Maryia Lu
  • Li Wei Rachel Tay
  • Jingquan He
  • Guangwei Du
Part of the Methods in Molecular Biology book series (MIMB, volume 1406)

Abstract

Phospholipids are important signaling molecules that regulate cell proliferation, death, migration, and metabolism. Many phospholipid signaling cascades are altered in breast cancer. To understand the functions of phospholipid signaling molecules, genetically encoded phospholipid biosensors have been developed to monitor their spatiotemporal dynamics. Compared to other phospholipids, much less is known about the subcellular production and cellular functions of phosphatidic acid (PA), partially due to the lack of a specific and sensitive PA biosensor in the past. This chapter describes the use of a newly developed PA biosensor, PASS, in two applications: regular fluorescent microscopy and fluorescence lifetime imaging microscopy-Förster/fluorescence resonance energy transfer (FLIM-FRET). These protocols can be also used with other phospholipid biosensors.

Key words

Phospholipid Phosphatidic acid PASS Cancer Immunofluorescence Fluorescence lifetime imaging microscopy FLIM Fluorescence resonance energy transfer FRET 

Notes

Acknowledgements

This work was supported by a research grant RP130425 from the Cancer Prevention and Research Institute of Texas (CPRIT) and a research grant R01HL119478 from the National Heart, Lung, and Blood Institute of the National Institutes of Health to GD, and a UTHealth Innovation for Cancer Prevention Research Training Program Predoctoral Fellowship grant RP140103 from the CPRIT to ML. The content is solely the responsibility of the authors and does not necessarily represent the official views of the CPRIT and National Institutes of Health.

References

  1. 1.
    Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112):651–657PubMedCrossRefGoogle Scholar
  2. 2.
    Maekawa M, Fairn GD (2014) Molecular probes to visualize the location, organization and dynamics of lipids. J Cell Sci 127(22):4801–4812PubMedCrossRefGoogle Scholar
  3. 3.
    Wymann MP, Schneiter R (2008) Lipid signalling in disease. Nat Rev Mol Cell Biol 9(2):162–176PubMedCrossRefGoogle Scholar
  4. 4.
    Park JB, Lee CS, Jang JH, Ghim J, Kim YJ, You S, Hwang D, Suh PG, Ryu SH (2012) Phospholipase signalling networks in cancer. Nat Rev Cancer 12(11):782–792PubMedCrossRefGoogle Scholar
  5. 5.
    Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10(8):789–799PubMedCrossRefGoogle Scholar
  6. 6.
    van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9(2):112–124PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Rusten TE, Stenmark H (2006) Analyzing phosphoinositides and their interacting proteins. Nat Methods 3(4):251–258PubMedCrossRefGoogle Scholar
  8. 8.
    Balla T, Varnai P (2002) Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci STKE 2002(125):pl3PubMedGoogle Scholar
  9. 9.
    Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9(2):99–111PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang Y, Du G (2009) Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases. Biochim Biophys Acta 1791(9):850–855PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Peng X, Frohman MA (2012) Mammalian phospholipase D physiological and pathological roles. Acta Physiol (Oxf) 204(2):219–226CrossRefGoogle Scholar
  12. 12.
    Shulga YV, Topham MK, Epand RM (2011) Regulation and functions of diacylglycerol kinases. Chem Rev 111(10):6186–6208PubMedCrossRefGoogle Scholar
  13. 13.
    Dominguez CL, Floyd DH, Xiao A, Mullins GR, Kefas BA, Xin W, Yacur MN, Abounader R, Lee JK, Wilson GM, Harris TE, Purow BW (2013) Diacylglycerol kinase alpha is a critical signaling node and novel therapeutic target in glioblastoma and other cancers. Cancer Discov 3(7):782–797PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Bruntz RC, Lindsley CW, Brown HA (2014) Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer. Pharmacol Rev 66(4):1033–1079PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Gomez-Cambronero J (2014) Phospholipase D in cell signaling: from a myriad of cell functions to cancer growth and metastasis. J Biol Chem 289(33):22557–22566PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Zhang F, Wang Z, Lu M, Yonekubo Y, Liang X, Zhang Y, Wu P, Zhou Y, Grinstein S, Hancock JF, Du G (2014) Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells. Mol Cell Biol 34(1):84–95PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Lasserre R, Guo XJ, Conchonaud F, Hamon Y, Hawchar O, Bernard AM, Soudja SM, Lenne PF, Rigneault H, Olive D, Bismuth G, Nunes JA, Payrastre B, Marguet D, He HT (2008) Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation. Nat Chem Biol 4(9):538–547PubMedCrossRefGoogle Scholar
  18. 18.
    Simons K, Gerl MJ (2010) Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11(10):688–699PubMedCrossRefGoogle Scholar
  19. 19.
    Harding AS, Hancock JF (2008) Using plasma membrane nanoclusters to build better signaling circuits. Trends Cell Biol 18(8):364–371PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Ariotti N, Liang H, Xu Y, Zhang Y, Yonekubo Y, Inder K, Du G, Parton RG, Hancock JF, Plowman SJ (2010) Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation. Mol Cell Biol 30(15):3795–3804PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Zhou Y, Liang H, Rodkey T, Ariotti N, Parton RG, Hancock JF (2014) Signal integration by lipid-mediated spatial cross talk between Ras nanoclusters. Mol Cell Biol 34(5):862–876PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Levitt JA, Matthews DR, Ameer-Beg SM, Suhling K (2009) Fluorescence lifetime and polarization-resolved imaging in cell biology. Curr Opin Biotechnol 20(1):28–36PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Maryia Lu
    • 1
  • Li Wei Rachel Tay
    • 1
  • Jingquan He
    • 1
  • Guangwei Du
    • 1
  1. 1.Department of Integrative Biology and PharmacologyThe University of Texas Health Science Center at HoustonHoustonUSA

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