Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse.
Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.
Evergren
Emma
E
Laboratory of Neuronal Membrane Trafficking, Center of Excellence in Developmental Biology, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Marcucci
Melissa
M
Tomilin
Nikolay
N
Löw
Peter
P
Slepnev
Vladimir
V
Andersson
Fredrik
F
Gad
Helge
H
Brodin
Lennart
L
De Camilli
Pietro
P
Shupliakov
Oleg
O
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002490:Central Nervous System / Q000378:metabolism; D002846:Chromatography, Affinity; D002966:Clathrin / Q000737:chemistry*; D004705:Endocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007798:Lampreys; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000502:physiology*; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000378:metabolism*; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D002966:Clathrin; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C075637:amphiphysin; D005982:Glutathione Transferase