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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / metabolism*
  • Calcium Channels / metabolism*
  • Calcium Signaling*
  • Cell Membrane Permeability
  • Cells, Cultured
  • Coloring Agents / pharmacokinetics*
  • Endocytosis
  • Lipid Bilayers
  • Mice


  • Calcium Channels
  • Coloring Agents
  • Lipid Bilayers