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Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21960-5. doi: 10.1073/pnas.0909109106. Epub 2009 Dec 9.

FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes.

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  • 1Institut National de la Santé et de la Recherche Médicale, INSERM U603, Centre National de la Recherche Scientifique, UMR 8154, Laboratoire de Neurophysiologie & Nouvelles Microscopies, Université Paris Descartes, 45 Rue des Saints Pères, F-75006 Paris, France.

Abstract

The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca(2+)) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca(2+) homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca(2+) entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca(2+) for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca(2+) from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence.

PMID:
20007370
[PubMed - indexed for MEDLINE]
PMCID:
PMC2799853
Free PMC Article
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