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 th e 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 biologist s. 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 phospholi pid membranes. First, addition of physiological concentrations of PI(4,5)P- 2 (2%) to lipid Vesicles formed from mixtures of phosphatidylcholine (PC) a nd phosphatidylserine (PS) enhances the binding of labeled neomycin signifi cantly (40-fold for 5:1 PC/PS vesicles). Second, physiological concentratio ns of inositol-1,4,5-trisphosphate (10 mu M I(1,4,5)P-3) cause little trans location 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 translocat ion of the green fluorescent protein/phospholipase C-delta pleckstrin homol ogy (GFP-PH) constructs from membranes (Hirose et al., Science, 284 (1999) 1527). Third, fluorescence microscopy observations confirm that one can dis tinguish between PC/PS vesicles containing either 0 or 2% PI(4,5)P-2 by exp osing a mixture of the vesicles to labeled neomycin. Thus fluorescently lab eled neomycin could complement GFP-PH constructs to investigate the locatio n of PI(4,5)P-2 in cell membranes. (C) 2000 Elsevier Science B.V. All right s reserved.