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Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones
Author(s) -
LinaresClemente Pedro,
Rozas José L.,
Mircheski Josif,
GarcíaJuncoClemente Pablo,
MartínezLópez José A.,
NietoGonzález José L.,
Vázquez M. Eugenio,
Pintado C. Oscar,
FernándezChacón Rafael
Publication year - 2015
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp270112
Subject(s) - dynamin , endocytosis , exocytosis , bulk endocytosis , synaptic vesicle , microbiology and biotechnology , synaptic vesicle recycling , biology , chemistry , biophysics , vesicle , biochemistry , receptor mediated endocytosis , membrane , cell
Key points 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.Abstract 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.

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