Spatial and Temporal Aspects of Phosphoinositides in Endocytosis Studied in the Isolated Plasma Membranes

  • Ira MilosevicEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1847)


Endocytosis is a well-orchestrated cascade of lipid–protein and protein–protein interactions resulting in formation and internalization of vesicles. Membrane phospholipids have key regulatory functions in endocytosis and membrane traffic. I have previously described an in vitro assay based on the isolated, substrate-attached plasma membrane to study the spatial distribution and levels of phosphoinositides, in particular phosphatidylinositol-4,5-bisphospate [PI(4,5)P2]. This assay utilizes cultured cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membrane sheets with elements of cell cytoskeleton. Here, I describe an experimental procedure for “on-stage” and “off-stage” detection of PI(4,5)P2 spatial distribution, and semi-quantification of PI(4,5)P2 levels in the plasma membrane using fluorescence microscopy. Depending on the probe selected for lipid detection, this simple assay can be modified to study other plasmalemmal phospholipids and/or proteins.

Key words

Endocytosis Phospholipids Plasma membrane Cell-free assay Isolated membrane sheets Phosphoinositides PI(4,5)P2 Lipid probe 



I thank Dr. A. Milosevic and Dr. M. Barszczewski for kind help with the figures, and Dr. N. Raimundo for comments. The author declares no competing financial interests. This work is supported by the grants of the German Research Foundation (DFG) through the collaborative research center SFB-889 (project A8) and SFB-1190 (project P02), and the Emmy Noether Young Investigator Award (1702/1).


  1. 1.
    Kononenko NL, Haucke V (2015) Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85(3):484–496CrossRefGoogle Scholar
  2. 2.
    McMahon HT, Boucrot E (2012) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12(8):517–533CrossRefGoogle Scholar
  3. 3.
    Saheki Y, De Camilli P (2012) Synaptic vesicle endocytosis. Cold Spring Harb Perspect Biol 4(9):a005645CrossRefGoogle Scholar
  4. 4.
    Milosevic I. (2018) Revisiting the Role of Clathrin-Mediated Endoytosis in Synaptic Vesicle Recycling. Front Cell Neurosci.12:27.
  5. 5.
    Watanabe S, Boucrot E (2017) Fast and ultrafast endocytosis. Curr Opin Cell Biol 47:64–71CrossRefGoogle Scholar
  6. 6.
    Cremona O, De Camilli P (2001) Phosphoinositides in membrane traffic at the synapse. J Cell Sci 114(Pt 6):1041–1052PubMedGoogle Scholar
  7. 7.
    De Craene JO, Bertazzi DL, Bär S, Friant S (2017) Phosphoinositides, major actors in membrane trafficking and lipid Signaling pathways. Int J Mol Sci 18(3):E634CrossRefGoogle Scholar
  8. 8.
    Idevall-Hagren O, De Camilli P (2015) Detection and manipulation of phosphoinositides. Biochim Biophys Acta 1851(6):736–745CrossRefGoogle Scholar
  9. 9.
    Posor Y, Eichhorn-Grünig M, Haucke V (2015) Phosphoinositides in endocytosis. Biochim Biophys Acta. 1851(6):794–804. Epub 2014 Sep 28. Review. PMID: 25264171CrossRefGoogle Scholar
  10. 10.
    Vicinanza M, D’Angelo G, Di Campli A, De Matteis MA (2008) Function and dysfunction of the PI system in membrane trafficking. EMBO J 27(19):2457–2470CrossRefGoogle Scholar
  11. 11.
    Wenk MR, De Camilli P (2004) Protein–lipid interactions and phosphoinositide metabolism in membrane traffic: insights from vesicle recycling in nerve terminals. Proc Natl Acad Sci U S A 101:8262–8269CrossRefGoogle Scholar
  12. 12.
    de Wit H, Walter A, Milosevic I, Gulyás-Kovács A, Sørensen JB, Verhage M (2009) Four proteins that dock secretory vesicles to the target membrane. Cell 138(5):935–946CrossRefGoogle Scholar
  13. 13.
    Lang T, Bruns D, Wenzel D, Riedel D, Holroyd P, Thiele C, Jahn R (2001) SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J 20:2202–2213CrossRefGoogle Scholar
  14. 14.
    Milosevic I, Sørensen JB, Lang T, Krauss M, Nagy G, Haucke V, Jahn R, Neher E (2005) Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J Neurosci 25(10):2557–2565CrossRefGoogle Scholar
  15. 15.
    Nagy G, Milosevic I, Fasshauer D, Müller M, de Groot B, Lang T, Wilson MC, Sørensen JB (2005) Alternative splicing of SNAP-25 regulates secretion through non-conservative substitutions in the SNARE domain. Mol Biol Cell 16:5675–5685CrossRefGoogle Scholar
  16. 16.
    Nagy G, Milosevic I, Mohrmann R, Wiederhold K, Walter AM, Sørensen JB (2008) The SNAP-25 linker as an adaptation toward fast exocytosis. Mol Biol Cell 19(9):3769–3781CrossRefGoogle Scholar
  17. 17.
    Wu M, Huang B, Graham M, Raimondi A, Heuser JE, Zhuang X, De Camilli P (2010) Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system. Nat Cell Biol 12(9):902–908CrossRefGoogle Scholar
  18. 18.
    Avery J, Ellis DJ, Lang T, Holroyd P, Riedel D, Henderson RM, Edwardson JM, Jahn R (2000) A cell-free system for regulated exocytosis in PC12 cells. J Cell Biol 148:317–324CrossRefGoogle Scholar
  19. 19.
    Wu M, De Camilli P (2012) Supported native plasma membranes as platforms for the reconstitution and visualization of endocytic membrane budding. Methods Cell Biol 108:3–18PubMedGoogle Scholar
  20. 20.
    Sørensen JB, Nagy G, Varoqueaux F, Nehring RB, Brose N, Wilson MC, Neher E (2003) Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114(1):75–86CrossRefGoogle Scholar
  21. 21.
    Sørensen JB, Wiederhold K, Müller M, Milosevic I, Nagy G, de Groot B, Grubmüller H, Fasshauer D (2006) Sequential N- to C-terminal “zipping-up” of the SNARE complex drives priming and fusion of secretory vesicles. EMBO J 25(5):955–966CrossRefGoogle Scholar
  22. 22.
    Smith GL, Miller DJ (1985) Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent ions including calcium. Biochim Biophys Acta 839(3):287–299CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.European Neuroscience Institute (ENI) and University Medical Center Göttingen (UMG)GöttingenGermany

Personalised recommendations