Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

Abstract

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture . Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma . In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation . Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective . Within the first few seconds after stimulation, relatively large vacuoles (-0.1 um) pinched off from the plasma membrane, both near to and far away from the active zones . Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation . The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles . This process was manifest first by the formation of relatively small (-0.05 wm) indentations in the plasma membrane, which occurred everywhere except at the active zones . These indentations first appeared at 1 s, reached a peak abundance of 5.5/,m2 by 30 s after the stimulus, and disappeared almost completely by 90 s . Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially . These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics : (a) the single endocytotic event is short lived relative to the time scale of two minutes ; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on .The discovery of synaptic vesicles (27,30,33,34,38) and the evidence for the quantal release of transmitter (5) led to the hypothesis that transmitter release is accomplished by the exocytosis of synaptic vesicles (6). Electron microscopy of chemically-fixed nerve terminals revealed pockets in the presynaptic membranes suggestive of exocytosis (9, 22, 31), but it was not possible to make temporal and quantitative comparisons between these structures and the number of transmitter quanta released .A quick-freezing machine, based on earlier designs by Van Harreveld (40-42) was developed specifically so that these

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

“…With respect to DYNAMIN, which is known to complete vesicle formation through membrane fission (44), previous studies have shown it is also present and functionally important at early stages of CLATHRIN-mediated endocytosis (45,46). AZ localization of endocytic proteins might suggest a mechanism of release site clearance involving rapid formation of CLATHRIN-coated vesicles directly from the AZ; however, this mechanism is not favored, primarily because ultrastructural studies indicate that CLATHRIN-coated vesicles form at PAZ, rather than AZ, regions of the plasma membrane (47,48) (Fig. 4B, iii).…”

“…4). Particle clusters were thought to dissipate after vesicle fusion (51), and further analysis suggested they may reassemble within CLATHRIN-coated pits at the PAZ (47). In contrast, recent superresolution light microscopy studies showed that native synaptic vesicle proteins deposited in the plasma membrane from different vesicles do not mix before endocytosis but, instead, remain in distinct clusters and are retrieved separately (53).…”

α-Synuclein – Regulator of Exocytosis, Endocytosis, or Both?

“…There are four major modes of endocytosis to differentiate, referred to as: kiss-and-run of synaptic vesicles, estimated to occur in <1 s time-frame [51]; clathrin-mediated endocytosis (CME), occurring in a 10-20 s time-frame [52][53][54][55]; ultrafast endocytosis followed by endosomal budding (initial membrane retrieval 100 ms, vesicle recovery 5-6 s) [56]; and bulk endocytosis [57]. Mechanisms of clathrin-mediated endocytosis are well established and many key players are known today; however, mechanisms for the kiss-and-run mode are under sustained debate and a molecular identity involved in the process is still missing.…”

Synaptic vesicle recycling in synapsin I knock-out mice.

“…The recycling is accomplished in less than 1 min by a series of steps beginning with a very efficient endocytic retrieval of synaptic vesicle components and culminating with the regeneration of releasable synaptic vesicles (Heuser and Reese, 1973;Miller and Heuser, 1984;Valtorta et al, 1988;Ryan and Smith, 1995; for reviews see Betz and Wu, 1995;De Camilli and Takei, 1996).…”