Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation  Natalia L. Kononenko, Volker Haucke  Neuron  Volume 85, Issue 3, Pages 484-496 (February 2015) DOI: 10.1016/j.neuron.2014.12.016 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Electron Microscopy of Recycling SV Membranes (A) Electron micrograph of a large axonal process (PRSN) of the earthworm, containing numerous synaptic vesicles (SVs). PSN, portion of another axon; ER, endoplasmic reticulum; Y, a specialized synaptic contact. Reproduced with permission from De Robertis and Bennett (1955). (B) SV recycling at the lobster neuromuscular junction, by exposing the synapse to HRP during stimulation for three periods of 10 min each, with 16,000 stimuli in total. HRP is present within many small vesicles in an axon ending, indicated by arrows. Scale bar represents 0.5 μm. Reproduced with permission from Holtzman et al. (1971). (C) An example of HRP retained within bulk structures (cisternae, C) at the frog neuromuscular junction stimulated constantly for 15 min and fixed immediately after. Reproduced with permission from Heuser and Reese (1973). (D) Original drawings by Heuser and Reese (1973), illustrating a progressive depletion of synaptic vesicles (open circles) and appearance ofcisternae (filled in black) in frog nerve terminal stimulated at 10 Hz for 1 and 15 min. The numbers indicate the area per section of synaptic vesicle membrane + cisternal membrane + surface membrane = total membrane. Reproduced with permission from Heuser and Reese (1973). (E) Clathrin-coated pits and clathrin-coated vesicles observed in the vicinity of the plasma membrane at a frog nerve terminal stimulated at 10 Hz for 1 min. Reproduced with permission from Heuser and Reese (1973). (F) Uncoated Ω-shaped intermediates at the active zone of frog nerve terminals stimulated at 2 Hz for 2 hr. Reproduced with permission from Ceccarelli et al. (1972). Neuron 2015 85, 484-496DOI: (10.1016/j.neuron.2014.12.016) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Current Models of SV Membrane Retrieval (A) Kiss-and-run mode. Opening of a fusion pore is sufficient for discharge of SV content. After closure of the pore vesicles detach from the plasma membrane and are directly reused through a fast mechanism. (B) “Classical” CME. Opening of a fusion pore is followed by complete SV fusion with the plasma membrane. SV components are subsequently retrieved directly from the cell surface through the formation of a clathrin-coated vesicle, which following uncoating, reenters the SV reserve pool. This mechanism operates on a timescale of tens of seconds. (C) Ultrafast mode. Fast retrieval of SV membranes after single or more APs occurs by membrane invaginations corresponding to approximately 4 SVs formed laterally to the active zone. SVs may be regenerated primarily from the resulting primary endocytic vesicles or from endosome-like vacuoles (ELVs) through a slow, presumably clathrin-dependent mechanism. (D) Strong tetanic or chemical stimulations cause the appearance of large bulk endocytic structures, which are subsequently resolved into SVs via clathrin-dependent and/or clathrin-independent mechanisms. Neuron 2015 85, 484-496DOI: (10.1016/j.neuron.2014.12.016) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Hypothetical Model for SV Membrane Retrieval and Reformation Left: high activity firing induces a steep rise in presynaptic calcium levels and triggers fast membrane retrieval via endosome-like vacuoles (ELVs) upstream of clathrin coat assembly. ELVs are subsequently converted into SVs via clathrin/AP-2-mediated budding and possibly additional pathways (i.e., via AP-3). Right: under conditions of low-frequency activity presynaptic calcium levels remain comparably low and membrane fission is rate limiting. This allows clathrin/AP-2 coats to assemble prior to membrane fission, resulting in SV reformation directly from the plasma membrane. Neuron 2015 85, 484-496DOI: (10.1016/j.neuron.2014.12.016) Copyright © 2015 Elsevier Inc. Terms and Conditions