FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes.
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.
Li
Dongdong
D
Institut 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.
Hérault
Karine
K
Oheim
Martin
M
Ropert
Nicole
N
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004396:Coloring Agents / Q000493:pharmacokinetics*; D004705:Endocytosis; D008051:Lipid Bilayers; D051379:Mice
D015220:Calcium Channels; D004396:Coloring Agents; D008051:Lipid Bilayers