Fluorescently labeled neomycin as a probe of phosphatidylinositol-4,5-bisphosphate in membranes

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2), 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)P-2 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)P-2 in model phospholipid membranes. First, addition of physiological concentrations of PI(4,5)P-2 (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 mu M I(1,4,5)P-3) cause little translocation of neomycin from PC/PS/PI(4,5)P-2 membranes to the aqueous phase, whereas the same concentrations of I(1,4,5)P-3 cause significant translocation of the green fluorescent protein/phospholipase C-delta 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)P-2 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)P-2 in cell membranes. (C) 2000 Elsevier Science B.V. All rights reserved.