title: Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones.
abstract: CONCLUSIONS
  Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction.
  Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB.
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Linares-Clemente
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: José L
authors.initials: JL
authors.lastname: Rozas
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: Josif
authors.initials: J
authors.lastname: Mircheski
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: García-Junco-Clemente
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Martínez-López
authors.forename: José L
authors.initials: JL
authors.lastname: Nieto-González
authors.affiliation: Departamento Química Orgánica y Centro Singular de Investigación en Química Biolóxica y Materiais Moleculares (CIQUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: M Eugenio
authors.initials: ME
authors.lastname: Vázquez
authors.affiliation: Centro Producción y Experimentación Animal, Universidad de Sevilla, Seville, Spain.
authors.forename: C Oscar
authors.initials: CO
authors.lastname: Pintado
authors.affiliation: Instituto de Biomedicina de Sevilla, IBiS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, and CIBERNED, Seville, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Fernández-Chacón
mesh_terms: D000818:Animals; D034281:Dynamins / Q000037:antagonists & inhibitors*; D004705:Endocytosis; D006835:Hydrazones / Q000494:pharmacology*; D051379:Mice; D009046:Motor Neurons / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology
chemical_list: D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D034281:Dynamins