Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.
The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.
Roux
Sylvie
S
Unité d'Embryologie Moléculaire, Institut Pasteur, Unités de Recherche Associées 2578, Centre National de la Recherche Scientifique, 25 rue du Dr Roux, 75724 Paris, France. sroux@nbcm..cnrs-gif.fr
Colasante
Cesare
C
Saint Cloment
Cécile
C
Barbier
Julien
J
Curie
Thomas
T
Girard
Emmanuelle
E
Molgó
Jordi
J
Brûlet
Philippe
P
D000818:Animals; D001370:Axonal Transport / Q000502:physiology; D005260:Female; D049452:Green Fluorescent Proteins / Q000493:pharmacokinetics*; D066298:In Vitro Techniques; D051379:Mice; D016253:Microscopy, Immunoelectron; D009046:Motor Neurons / Q000378:metabolism; D009411:Nerve Endings / Q000378:metabolism / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics; D013744:Tetanus Toxin / Q000493:pharmacokinetics*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D049452:Green Fluorescent Proteins