Assessment of cation trapping by cellular acidic compartments

Authors: Marceau, FrançoisRoy, CarolineBouthillier, Johanne
Edited by: P. Michael, Conn
Abstract: All nucleated cells, from yeast to animal cells, concentrate cationic chemicals (weak bases with a pKa ~8-10) into acidic cell compartments (low retro-diffusion under a protonated form at low pH = ion trapping). The proton pump vacuolar (V)-ATPase is the driving force of this pseudotransport that concerns acidic organelles (mainly late endosomes and lysosomes). The latter rapidly become swollen (osmotic vacuolization) and macroautophagic. Cation concentration in cells is not proven to involve membrane transporters, but is prevented or reversed by inhibitors of V-ATPase, such as bafilomycin A1. Lipophilicity is a major determinant of the apparent affinity of this pseudotransport because simple diffusion of the uncharged form supports it. Quinacrine is a formerly used anti-parasitic drug that is intensely fluorescent, lipophilic and a tertiary amine. The drug, at micromolar concentrations, is proposed as a superior probe for assessing cation trapping by cellular acidic compartments, being readily quantified using fluorometry in cell extracts and analyzed using microscopy and cytofluorometry (fluorescence settings for fluorescein being applicable). Further, cells respond to micromolar levels of quinacrine by autophagic accumulation (e.g., accumulation of the activated macroautophagic effector LC3 II, immunoblots), an objective and universal response to sequestered amines.
Document Type: Chapitre d'ouvrage
Issue Date: 17 December 2013
Open Access Date: Restricted access
Document version: AM
This document was published in: Endosome Signaling Part A. (Methods in Enzymology, Vol. 534, 119–131 (2014))
Academic Press
Alternative version: 10.1016/B978-0-12-397926-1.00007-X
Collection:Chapitres de livre

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