Abstract
Activity‐dependent bulk endocytosis generates synaptic vesicles (SVs) during intense neuronal activity via a two‐step process. First, bulk endosomes are formed direct from the plasma membrane from which SVs are then generated. SV generation from bulk endosomes requires the efflux of previously accumulated calcium and activation of the protein phosphatase calcineurin. However, it is still unknown how calcineurin mediates SV generation. We addressed this question using a series of acute interventions that decoupled the generation of SVs from bulk endosomes in rat primary neuronal culture. This was achieved by either disruption of protein–protein interactions via delivery of competitive peptides, or inhibition of enzyme activity by known inhibitors. SV generation was monitored using either a morphological horseradish peroxidase assay or an optical assay that monitors the replenishment of the reserve SV pool. We found that SV generation was inhibited by, (i) peptides that disrupt calcineurin interactions, (ii) an inhibitor of dynamin I GTPase activity and (iii) peptides that disrupt the phosphorylation‐dependent dynamin I–syndapin I interaction. Peptides that disrupted syndapin I interactions with eps15 homology domain‐containing proteins had no effect. This revealed that (i) calcineurin must be localized at bulk endosomes to mediate its effect, (ii) dynamin I GTPase activity is essential for SV fission and (iii) the calcineurin‐dependent interaction between dynamin I and syndapin I is essential for SV generation. We therefore propose that a calcineurin‐dependent dephosphorylation cascade that requires both dynamin I GTPase and syndapin I lipid‐deforming activity is essential for SV generation from bulk endosomes.
Highlights
Calcineurin, dynamin I and syndapin I were all present, suggesting they may perform parallel roles in synaptic vesicle (SV) generation at the bulk endosome. We demonstrate that both calcineurin–dynamin I and dynamin I– syndapin I interactions are essential for SV generation at bulk endosomes
Calcineurin localization at bulk endosomes is required to SV generation The release of accumulated extracellular calcium from bulk endosomes during their acidification is essential for SV generation from these organelles (Cheung and Cousin 2013)
This acidification-dependent calcium release activates the Dynamin and syndapin form SVs from endosomes 573 (c) calcium-dependent protein phosphatase calcineurin, an event which is essential for SV budding (Cheung and Cousin 2013)
Summary
Clathrinmediated endocytosis (CME) is triggered during mild periods of neuronal activity (Granseth et al 2006), this mode saturates during high bursts of high-frequency firing (Clayton et al 2008) During these specific periods, activity-dependent bulk endocytosis (ADBE) is the dominant endocytosis mode (Clayton et al 2008), forming bulk endosomes direct from the plasma membrane from which SVs are generated (Kokotos and Cousin 2015). Activity-dependent bulk endocytosis (ADBE) is the dominant endocytosis mode (Clayton et al 2008), forming bulk endosomes direct from the plasma membrane from which SVs are generated (Kokotos and Cousin 2015) Both ultrafast and CME refill the RRP (Granseth and Lagnado 2008; Watanabe et al 2014), whereas ADBE exclusively replenishes the reserve SV pool (Cheung et al 2010).
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