Confocal imaging and tracking of the exocytotic routes for D-serine-mediated gliotransmission.
D-Serine is an astrocyte-derived regulator for N-methyl-D-aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D-serine. We report that D-serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D-serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+-ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D-serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D-serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D-serine.
Martineau
Magalie
M
CNRS, Institut de Neurobiologie Alfred Fessard, FRC 2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040, F-91198 Gif-sur-Yvette, France.
Galli
Thierry
T
Baux
Gérard
G
Mothet
Jean-Pierre
JP
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D018613:Microscopy, Confocal / Q000379:methods; D009457:Neuroglia / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*
D015415:Biomarkers; D012694:Serine