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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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).
Kononenko NL, Haucke V (2015) Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85(3):484–496CrossRefGoogle Scholar
McMahon HT, Boucrot E (2012) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12(8):517–533CrossRefGoogle Scholar
Saheki Y, De Camilli P (2012) Synaptic vesicle endocytosis. Cold Spring Harb Perspect Biol 4(9):a005645CrossRefGoogle Scholar
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
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
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
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
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
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
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
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
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
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
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
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
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