Abstract
Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a minor component of the plasma membrane, is important in signal transduction, exocytosis, and ion channel activation. Thus fluorescent probes suitable for monitoring the PI(4,5)P2 distribution in living cells are valuable tools for cell biologists. We report here three experiments that show neomycin labeled with either fluorescein or coumarin can be used to detect PI(4,5)P2 in model phospholipid membranes. First, addition of physiological concentrations of PI(4,5)P2 (2%) to lipid vesicles formed from mixtures of phosphatidylcholine (PC) and phosphatidylserine (PS) enhances the binding of labeled neomycin significantly (40-fold for 5:1 PC/PS vesicles). Second, physiological concentrations of inositol-1,4,5-trisphosphate (10 μM I(1,4,5)P3) cause little translocation of neomycin from PC/PS/PI(4,5)P2 membranes to the aqueous phase, whereas the same concentrations of I(1,4,5)P3 cause significant translocation of the green fluorescent protein/phospholipase C-δ pleckstrin homology (GFP-PH) constructs from membranes (Hirose et al., Science, 284 (1999) 1527). Third, fluorescence microscopy observations confirm that one can distinguish between PC/PS vesicles containing either 0 or 2% PI(4,5)P2 by exposing a mixture of the vesicles to labeled neomycin. Thus fluorescently labeled neomycin could complement GFP-PH constructs to investigate the location of PI(4,5)P2 in cell membranes. Copyright (C) 2000 Elsevier Science B.V.
Original language | English |
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Pages (from-to) | 35-48 |
Number of pages | 14 |
Journal | Biochimica et Biophysica Acta - Biomembranes |
Volume | 1464 |
Issue number | 1 |
DOIs | |
State | Published - Mar 15 2000 |
Bibliographical note
Funding Information:This work was supported by NIH grant GM 24971 and NSF grant MCG 9729538 to SM, NIH grant NS 29632 to GP and NIH grant GM 50388 to AM.
Keywords
- Binding
- Fluorescence
- Microscopy
- Neomycin
- Phosphatidylinositol-4,5-bisphosphate
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Cell Biology