title: VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis.
abstract: The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Nicholson-Fish
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland.
authors.forename: Alexandros C
authors.initials: AC
authors.lastname: Kokotos
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Gillingwater
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland. Electronic address: k.smillie@ed.ac.uk.
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Smillie
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland. Electronic address: m.cousin@ed.ac.uk.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cousin
mesh_terms: D000200:Action Potentials / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004622:Embryo, Mammalian; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D006575:Heterocyclic Compounds, 3-Ring / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D008297:Male; D051379:Mice; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050496:Vesicular Glutamate Transport Proteins / Q000378:metabolism
chemical_list: D006575:Heterocyclic Compounds, 3-Ring; D050683:R-SNARE Proteins; D034741:RNA, Small Interfering; D012235:Rhodamines; C533192:VAMP4 protein, rat; D050496:Vesicular Glutamate Transport Proteins; C412715:tetramethylrosamine