Ultrafast Recycling of Synaptic Vesicles

Abstract

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...