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

• Published in: Biochimica et biophysica acta 2000-03, Vol.1464 (1), p.35-48
• Main Authors: Arbuzova, Anna, Martushova, Katherine, Hangyás-Mihályné, Gyöngyi, Morris, Andrew J., Ozaki, Shoichiro, Prestwich, Glenn D., McLaughlin, Stuart
• Format: Article
• Language: English
• Subjects: Acetates - chemistry; Binding; Cell Membrane - chemistry; Chromones - chemistry; Fluorescein-5-isothiocyanate - chemistry; Fluorescence; Fluorescent Dyes; Lipid Bilayers - chemistry; Microscopy; Microscopy, Fluorescence; Molecular Structure; Neomycin; Neomycin - chemistry; Phosphatidylinositol 4,5-Diphosphate - analysis; Phosphatidylinositol-4,5-bisphosphate; Phosphatidylinositols - analysis; Phospholipids - chemistry
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/S0005-2736(99)00243-6

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 μM 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-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)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.