Clathrin regenerates synaptic vesicles from endosomes

Abstract

SummaryUltrafast endocytosis can retrieve a single large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 seconds, which in turn become small-diameter synaptic vesicles 5-6 seconds after stimulation. We disrupted clathrin function using RNAi and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results explain in large part discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

Vesicular Glutamate Transporter 1 Orchestrates Recruitment of Other Synaptic Vesicle Cargo Proteins during Synaptic Vesicle Recycling

“…However, the demonstration that different cargos can be retrieved at the same terminals with significantly different kinetics suggests that different cargos are not equally sorted at the plasma membrane into the vesicles that bud from the plasma membrane, arguing that an additional endosomal sorting step(s) is(are) likely occurring. Such endocytic sorting for SVs has been suggested in recent high-pressure freeze experiments (27). The picture that emerges regarding endocytosis at synapses is considerably different from that for classical clathrin-mediated endocytosis.…”

Tissue-specific dynamin-1 deletion at the calyx of Held decreases short-term depression through a mechanism distinct from vesicle resupply

“…Indeed, previous studies of ultrastructure reported an increase in bulk endocytosis-a fast and efficient membrane retrieval mechanism primarily induced during very intense stimulation-in synapses lacking dynamin-1 (67). In addition, it was shown that actin plays an important role in bulk and ultrafast endocytosis (68)(69)(70)(71)(72). These observations raise the possibility that an actin-dependent increase in bulk endocytosis in cKO could make release site clearance more efficient than in control during high-frequency release.…”

“…7 E and G, Insets), indicating that both processes may be related through an actin-dependent mechanism. Dynamin can regulate actin function through direct and indirect interactions (65,82), and actin is important for bulk (68)(69)(70) and ultrafast endocytosis (72). Interestingly, dynamin KO leads to alteration of actin polymerization and reorganization in other types of cells (34,66).…”

σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila

“…Taken together, these data suggest that s 2 -adaptin facilitates the basal synaptic transmission at Drosophila NMJ synapses. The functional requirement of the AP2 complex in SV membrane retrieval at mammalian hippocampal synapses has been debated (Kononenko et al 2014;Watanabe et al 2014). Hence, in order to test the functional requirement of s 2 -adaptin in SV re-formation from the presynaptic membrane, we directly measured the rate of re-formation of normal-pH synaptic vesicles using synaptopHluorin imaging (Poskanzer et al 2006;Kumar et al 2009).…”

“…Taken together, these data suggest that all the subunits of AP2 along with phospholipid PI(4,5)P2 are obligate partners for regulating morphological plasticity at Drosophila NMJs. The AP2 complex is believed to be the major adapter for clathrin assembly during CME at the synapses and hence is believed to be essential for SV retrieval and regeneration of SVs for subsequent rounds of neurotransmitter release (Gonzalez-Gaitan and Jackle 1997; Collins et al 2002;Heerssen et al 2008;Cheung et al 2010;Gu et al 2013;Watanabe et al 2014). Hence, in order to assess the need for the AP2 complex in endocytosis, we loaded s 2 -adaptin mutant and control synaptic terminals with FM1-43.…”