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

Anna Arbuzova, Katherine Martushova, Gyöngyi Hangyás-Mihályné, Andrew J. Morris, Shoichiro Ozaki, Glenn D. Prestwich, Stuart McLaughlin

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

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 languageEnglish
Pages (from-to)35-48
Number of pages14
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1464
Issue number1
DOIs
StatePublished - 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

Fingerprint

Dive into the research topics of 'Fluorescently labeled neomycin as a probe of phosphatidylinositol-4,5-bisphosphate in membranes'. Together they form a unique fingerprint.

Cite this