EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.
EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.
Bouvier
David
D
Département de pathologie et biologie cellulaire and Groupe de recherche sur le système nerveux central, Université de Montréal, Montréal, QC, Canada.
Tremblay
Marie-Eve
ME
Riad
Mustapha
M
Corera
Amadou T
AT
Gingras
Diane
D
Horn
Katherine E
KE
Fotouhi
Maryam
M
Girard
Martine
M
Murai
Keith K
KK
Kennedy
Timothy E
TE
McPherson
Peter S
PS
Pasquale
Elena B
EB
Fon
Edward A
EA
Doucet
Guy
G
D000818:Animals; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004622:Embryo, Mammalian; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009388:Neostigmine / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000648:ultrastructure*; D011189:Potassium Chloride / Q000494:pharmacology; D036122:Receptor, EphA4 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D050857:Synaptotagmins / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000378:metabolism
D002966:Clathrin; C493454:Slc18a2 protein, rat; C118623:Slc32a1 protein, rat; D050598:Vesicular Glutamate Transport Protein 1; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D050857:Synaptotagmins; D009388:Neostigmine; D011189:Potassium Chloride; D036122:Receptor, EphA4