To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated ~20 s after fusion by the assembly of clathrin scaffolds or in ~1 s by the reversal of fusion pores via ‘kiss-and-run’ endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy – ‘flash-and-freeze’ electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs with 50-100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover it is 200-fold faster than clathrin-mediated endocytosis. It is likely that ‘ultrafast endocytosis’ is specialized to rapidly restore the surface area of the membrane.

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.

Munc13 is a multidomain protein of presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here, we use biophysical, biochemical, and electrophysiological approaches to demonstrate that the central C2B-domain of Munc13 functions as a Ca2+-regulator of short-term synaptic plasticity. The crystal structure of the C2B-domain revealed an unusual Ca2+-binding site with an amphipathic α-helix. This configuration confers onto the C2B-domain unique Ca2+-dependent phospholipid-binding properties favoring phosphatidylinositolphosphates. A mutation that inactivated Ca2+-dependent phospholipid binding to the C2B-domain did not alter neurotransmitter release evoked by isolated action potentials, but depressed release evoked by action potential trains. In contrast, a mutation that increased Ca2+-dependent phosphatidylinositolbisphosphate binding to the C2B-domain enhanced release evoked by isolated action potentials and by action potential trains. Our data suggest that during repeated action potentials, Ca2+- and phosphatidylinositolphosphate-binding to the Munc13 C2B-domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

Neurons can fire at extremely high rates. To sustain neurotransmission, synaptic vesicles must be recycled locally at synapses. Two models for synaptic vesicle endocytosis have been put forward based on the morphological studies in frog neuromuscular junctions. Heuser and Reese proposed that endocytosis occurs via a slow mechanism using clathrin scaffolds. Ceccarelli and his coworkers proposed a fast mechanism, kiss-and-run. Since then, many studies have sought to identify the mechanism for synaptic vesicle endocytosis. However, instead of resolving the issue, conflicting evidence has accumulated over the years. The molecular studies have suggested clathrin and clathrinassociated proteins are essential. However, the kinetics studies have suggested that both forms co-exist. Our data identify a third pathway that is fast, but requires clathrin to regenerate vesicles. To investigate how endocytosis takes place, we developed a method, 'flashand-freeze' fixation that couples optogenetic stimulation with rapid highpressure freezing and captures endocytosis at millisecond temporal resolution. To our surprise, vesicle membrane is recovered via ultrafast endocytosis within 100 ms following a single stimulus. The large endocytic vesicles then fuse to form an endosome and are resolved by clathrin into synaptic vesicles in 5-6 s. When experiments are performed at 20 C instead of 37 C, ultrafast endocytosis fails, and clathrin regenerates synaptic vesicles directly from plasma membrane. These results suggest that recycling of synaptic vesicles is normally a rapid two-step process: ultrafast endocytosis that removes excess membrane from the surface and then clathrin-mediated biogenesis of synaptic vesicles from endosomes. Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin (CPX) inhibits fusion through interactions between its highly conserved central helix and the SNARE complex. Two poorly conserved domains (the accessory helix and the C-terminal domain) on either side of the central helix (CH) are also required for inhibition of spontaneous fusion. We found that the C-terminal domain (CTD) binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to highly curved membranes such as SVs. Membrane curvature enhanced CPX binding and induced conformational changes in a critical amphipathic region of the CTD. The accessory helix (AH) of CPX contributes to the inhibition of exocytosis but the molecular mechanism for this function remains unknown. Several models have been proposed for the role of AH based on the concept that AH competes with VAMP for a binding site on the SNARE complex. Using a series of AH mutations and chimeras with mouse AH together with NMR and CD spectroscopy, electrophysiology, and behavioral assays, we identified key features of the AH and CH required for inhibition of SV f...