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Analysis of Phosphoinositide Dynamics During Phagocytosis Using Genetically Encoded Fluorescent Biosensors

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Autophagosome and Phagosome

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 445))

Summary

Phosphoinositide signaling is essential for successful phagocytosis. Phosphoinositides regulate processes such as actin assembly and the recruitment of molecular motors required for ingestion, as well as fusion events required for the maturation of the phagosome. Phosphoinositides not only serve as substrates for the generation of second messengers, but also function to anchor to the membrane cytosolic proteins that contain phosphoinositide-binding motifs. Conventional methods for the detection of phosphoinositides involve their extraction from the cells and separation by chromatographic procedures. These approaches are laborious and expensive and fail to provide spatio-temporal information, which is critical when analyzing localized and transient phenomena like phagocytosis. In this chapter we describe a method to monitor phosphoinositides dynamically by transfection of fluorescently tagged probes (biosensors) into cultured macrophages. These biosensors are based on the fusion of phosphoinositide-binding protein domains with fluorescent proteins. Some specifications for live cell imaging of such phosphoinositide-specific probes are also provided.

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References

  1. Yeung, T., Ozdamar, B., Paroutis, P. and Grinstein, S. (2006) Lipid metabolism and dynamics during phagocytosis. Curr. Opin. Cell Biol. 18, 429–437.

    Article  CAS  PubMed  Google Scholar 

  2. Stuart, L. M. and Ezekowitz, R. A. (2005) Phagocytosis: elegant complexity. Immunity 22, 539–550.

    Article  CAS  PubMed  Google Scholar 

  3. Vieira, O. V. et al. (2001) Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J. Cell Biol. 155, 19–25.

    Article  CAS  PubMed  Google Scholar 

  4. Vieira, O. V., Botelho, R. J. and Grinstein, S. (2002) Phagosome maturation: aging gracefully. Biochem. J. 366, 689–704.

    CAS  PubMed  Google Scholar 

  5. Halet, G. (2005) Imaging phosphoinositide dynamics using GFP-tagged protein domains. Biol. Cell 97, 501–518.

    Article  CAS  PubMed  Google Scholar 

  6. Martin, T. F. J. (1997) Phosphoinositides as spatial regulators of membrane traffic. Curr. Opin. Neurobiol. 7, 331–338.

    Article  CAS  PubMed  Google Scholar 

  7. Martin, T. F. J. (1998) Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu. Rev. Cell Dev. Biol. 14, 231–264.

    Article  CAS  PubMed  Google Scholar 

  8. Toker, A. (1998) The synthesis and cellular roles of phosphatidylinositol 4,5-bisphosphate. Curr. Opin. Cell Biol. 10, 254–261.

    Article  CAS  PubMed  Google Scholar 

  9. Payrastre, B., Missy, K., Giuriato, S., Bodin, S., Plantavid, M. and Gratacap, M. (2001) Phosphoinositides: key players in cell signaling, in time and space. Cell Signal. 13, 377–387.

    Article  CAS  PubMed  Google Scholar 

  10. Itoh, T. and Takenawa, T. (2002) Phosphoinositide-binding domains: Functional units for temporal and spatial regulation of intracellular signaling. Cell Signal. 14, 733–743.

    Article  CAS  PubMed  Google Scholar 

  11. Lemmon, M. A. (2003) Phosphoinositide recognition domains. Traffic 4, 201–213.

    Article  CAS  PubMed  Google Scholar 

  12. Botelho, R. J., Teruel, M., Dierckman, R., et al. (2000) Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell Biol. 151, 1353–1368.

    Article  CAS  PubMed  Google Scholar 

  13. Scott, C. C. et al. (2005) Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J. Cell Biol. 169, 139–149.

    Article  CAS  PubMed  Google Scholar 

  14. Marshall, J. G., Booth, J. W., Stambolic, V., et al. (2001) Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis. J. Cell Biol. 153, 1369–1380.

    Article  CAS  PubMed  Google Scholar 

  15. Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon, M. A. and Schlessinger, J. (1998) Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J. 17, 414–422.

    Article  CAS  PubMed  Google Scholar 

  16. Cox, D., Berg, J. S., Cammer, M., et al. (2002) Myosin X is a downstream effector of PI(3)K during phagocytosis. Nat. Cell Biol. 4, 469–477.

    CAS  PubMed  Google Scholar 

  17. Cox, D., Tseng, C. C., Bjekic, G. and Greenberg, S. (1999) A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J. Biol. Chem. 274, 1240–1247.

    Article  CAS  PubMed  Google Scholar 

  18. Ellson, C. D., Anderson, K. E., Morgan, G., et al. (2001) Phosphatidylinositol 3-phosphate is generated in phagosomal membranes. Curr. Biol. 11, 1631–1635.

    Article  CAS  PubMed  Google Scholar 

  19. Raiborg, C., Bremnes, B., Mehlum, A., et al. (2001) FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. J. Cell Sci. 114, 2255–2263.

    CAS  PubMed  Google Scholar 

  20. Serunian, L. A., Auger, K. R. and Cantley, L. C. (1991) Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol. 198, 78–87.

    Article  CAS  PubMed  Google Scholar 

  21. van Dongen, C. J., Zwiers, H. and Gispen, W. H. (1985) Microdetermination of phosphoinositides in a single extract. Anal. Biochem. 144, 104–109.

    Article  PubMed  Google Scholar 

  22. Wenk, M. R. et al. (2003) Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nat. Biotechnol. 21, 813–817.

    Article  CAS  PubMed  Google Scholar 

  23. Lemmon, M. A. and Ferguson, K. M. (2000) Signal-dependent membrane targeting by pleckstrin homology (PH) domains. Biochem. J. 350, 1–18.

    Article  CAS  PubMed  Google Scholar 

  24. Ellson, C. D., Andrews, S., Stephens, L. R. and Hawkins, P. T. (2002) The PX domain: a new phosphoinositide-binding module. J. Cell Sci. 115, 1099–1105.

    CAS  PubMed  Google Scholar 

  25. Itoh, T., Koshiba, S., Kigawa, T., Kikuchi, A., Yokoyama, S. and Takenawa, T. (2001) Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 291, 1047–1051.

    Article  CAS  PubMed  Google Scholar 

  26. Gillooly, D. J., Simonsen, A. and Stenmark, H. (2001) Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins. Biochem. J. 355, 249–258.

    Article  CAS  PubMed  Google Scholar 

  27. Hamada, K., Shimizu, T., Matsui, T., Tsukita, S. and Hakoshima, T. (2000) Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J. 19, 4449–4462.

    Article  CAS  PubMed  Google Scholar 

  28. Doerks, T., Strauss, M., Brendel, M. and Bork, P. (2000) GRAM, a novel domain in glucosyltransferases, myotubularins and other putative membrane-associated proteins. Trends Biochem. Sci. 25, 483–485.

    Article  CAS  PubMed  Google Scholar 

  29. Levine, T. P. and Munro, S. (2002) Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695–704.

    Article  CAS  PubMed  Google Scholar 

  30. Balla, A., Tuymetova, G., Tsiomenko, A., Varnai, P. and Balla, T. (2005) A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1. Mol. Biol. Cell. 16, 1282–1295.

    Article  CAS  PubMed  Google Scholar 

  31. Godi, A. et al. (2004) FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nat. Cell Biol. 6, 393–404.

    Article  CAS  PubMed  Google Scholar 

  32. Yu, J. W. et al. (2004) Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell. 13, 677–688.

    Article  CAS  PubMed  Google Scholar 

  33. Roy, A. and Levine, T. P. (2004) Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p. J Biol. Chem. 279, 44683–44689.

    Article  CAS  PubMed  Google Scholar 

  34. Levine, T. P. and Munro, S. (1998) The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes. Curr. Biol. 8, 729–739.

    Article  CAS  PubMed  Google Scholar 

  35. Gillooly, D. J., Morrow, I. C., Lindsay, M., et al. (2000) Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588.

    Article  CAS  PubMed  Google Scholar 

  36. Ellson, C. D. et al. (2001) PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nat. Cell Biol. 3, 679–682.

    Article  CAS  PubMed  Google Scholar 

  37. Stauffer, T. P., Ahn, S. and Meyer, T. (1998) Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr. Biol. 8, 343–346.

    Article  CAS  PubMed  Google Scholar 

  38. Varnai, P. and Balla, T. (1998) Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-3H.inositol-labeled phosphoinositide pools. J. Cell Biol. 143, 501–510.

    Article  CAS  PubMed  Google Scholar 

  39. Santagata, S., Boggon, T. J., Baird, C. L., et al. (2001) G-protein signaling through tubby proteins. Science 292, 2041–2050.

    Article  CAS  PubMed  Google Scholar 

  40. Kimber, W. A. et al. (2002) Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem. J. 361, 525–536.

    Article  CAS  PubMed  Google Scholar 

  41. Dove, S. K. et al. (2004) Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors. EMBO J. 23, 1922–1933.

    Article  CAS  PubMed  Google Scholar 

  42. Dowler, S., Currie, R. A., Campbell, D. G., et al. (2000) Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem. J. 351, 19–31.

    Article  CAS  PubMed  Google Scholar 

  43. Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat , J. W. and Bourne, H. R. (2000) Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 287, 1037–1040.

    Article  CAS  PubMed  Google Scholar 

  44. Watton, S. J. and Downward, J. (1999) Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9, 433–436.

    Article  CAS  PubMed  Google Scholar 

  45. Komander, D. et al. (2004) Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates. EMBO J. 23, 3918–3928.

    Article  CAS  PubMed  Google Scholar 

  46. Dormann, D., Weijer, G., Parent, C. A., Devreotes, P. N. and Weijer, C. J. (2002) Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development. Curr. Biol. 12, 1178–1188.

    Article  CAS  PubMed  Google Scholar 

  47. Klarlund, J. K., Tsiaras, W., Holik, J. J., Chawla, A. and Czech, M. P. (2000) Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs. J. Biol. Chem. 275, 32816–32821.

    Article  CAS  PubMed  Google Scholar 

  48. Venkateswarlu, K., Oatey, P. B., Tavare, J. M. and Cullen, P. J. (1998) Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr. Biol. 8, 463–466.

    Article  CAS  PubMed  Google Scholar 

  49. Venkateswarlu, K., Gunn-Moore, F., Oatey, P. B., Tavare, J. M. and Cullen, P. J. (1998) Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain. Biochem. J. 335, 139–146.

    CAS  PubMed  Google Scholar 

  50. Varnai, P., Rother, K. I. and Balla, T. (1999) Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton’s tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274, 10983–10989.

    Article  CAS  PubMed  Google Scholar 

  51. Nagel, W., Schilcher, P., Zeitlmann, L. and Kolanus, W. (1998) The PH domain and the polybasic c domain of cytohesin-1 cooperate specifically in plasma membrane association and cellular function. Mol. Biol. Cell. 9, 1981–1994.

    CAS  PubMed  Google Scholar 

  52. Venkateswarlu, K., Gunn-Moore, F., Tavare, J. M. and Cullen, P. J. (1999) EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain. J. Cell Sci. 112, 1957–1965.

    CAS  PubMed  Google Scholar 

  53. Varnai, P. and Balla, T. (2006) Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. Biochim. Biophys. Acta. 1761, 957–967.

    CAS  PubMed  Google Scholar 

  54. Graf, R., Rietdorf, J. and Zimmermann, T. (2005) Live cell spinning disk microscopy. Adv. Biochem. Eng. Biotechnol. 95, 57–75.

    PubMed  Google Scholar 

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Acknowledgments

Original work in the authors’ laboratory is supported by the Heart and Stroke Foundation of Ontario, the Canadian Cystic Fibrosis Foundation, and the Canadian Institutes for Health Research. G.C. is a fellow of the McLaughlin Centre for Molecular Medicine, and S.G. is the current holder of the Pitblado Chair in Cell Biology.

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Authors and Affiliations

  1. Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada

    Gabriela Cosío

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  1. Gabriela Cosío
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  2. Sergio Grinstein
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Editors and Affiliations

  1. Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Vojo Deretic

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Cosío, G., Grinstein, S. (2008). Analysis of Phosphoinositide Dynamics During Phagocytosis Using Genetically Encoded Fluorescent Biosensors. In: Deretic, V. (eds) Autophagosome and Phagosome. Methods in Molecular Biology™, vol 445. Humana Press. https://doi.org/10.1007/978-1-59745-157-4_18

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  • DOI: https://doi.org/10.1007/978-1-59745-157-4_18

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-853-9

  • Online ISBN: 978-1-59745-157-4

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