Synaptic vesicle life cycle

The life cycle of a SV is illustrated in Figure 8. During exocytosis neurotransmitter filled vesicles are mobilized to dock at the plasma membrane, where they undergo a priming process in which they become fusion competent. A calcium-signal then triggers vesicle fusion with the

Figure 8. The life cycle of a synaptic vesicle.

plasma membrane and the neurotransmitter is released into the synaptic cleft (reviewed in (Li and Chin, 2003). Following exocytosis, synaptic vesicle membranes and protein constituents are retrieved by endocytosis, sorted and loaded with neurotransmitter becoming mature synaptic vesicles. A major pathway for the retrieval of fused synaptic vesicles is clathrin-mediated endocytosis. This process requires vesicle membrane to be targeted for internalization through the recruitment of clathrin adaptors (AP2 and AP180) which in turn bind clathrin, resulting in formation of a clathrin-coated bud, fission of the coated vesicle, uncoating and sorting back to the vesicle pool (reviewed in (Harris et al., 2001; Richmond, 2005;

Richmond and Broadie, 2002).

Docking of SVs represents a reversible step of membrane association prior to the priming of the readily releasable pool of SVs, which are mobilised from the disparate regions of the terminal to the active zone preceding the release (Rizzoli and Betz, 2004). The mechanism which designates which one of the SVs from the active zone are docked, primed and released is still unknown, but a lot of efforts are invested in the definition of the functional relevance of vesicle positioning using techniques like high pressure freeze electron microscopy (EM), immunoEM and analyses of mutants of proteins known to be involved in docking and priming (Rostaing et al., 2004). Moreover, the specific docking molecular processes are not well understood (Robinson and Martin, 1998). A central player in the docking process seems to be RAB-3, a small GTPase that associates with SVs in C. elegans (Nonet et al., 1997). rab-3 mutants are only mildly uncoordinated and exhibit SVs dispersal at synapses. RAB-3 binds to Rim/UNC-10, a component of the active zone, and thus establishes a link between SVs and active zone cytomatrix. A similar reduction of docked vesicles at the active zone is found in unc-18 mutants, suggesting that UNC-unc-18 acts directly or indirectly as a facilitator of vesicle docking (Weimer and Richmond, 2005; Weimer et al., 2003). UNC-18 interacts with UNC-64 (ortholog of syntaxin, a membrane-anchored protein involved in SV fusion)(Saifee et al., 1998).

Priming refers to the molecular events following vesicle docking that lead to vesicle fusion competence. Priming appears to involve the formation of trans-SNARE complexes, in which the synaptic vesicle protein synaptobrevin (also called VAMP) forms a helical bundle with the plasma membrane proteins UNC-64/syntaxin and RIC-4/SNAP-25, which brings the SV and the plasma membrane in juxtaposition (Chen and Scheller, 2001). SNARE proteins are essential for C. elegans viability, as their mutants arrest at the L1 larval stage, limiting the characterization of the synaptic phenotype (Nonet et al., 1998; Saifee et al., 1998). Neuronal SNAREs interact with a number of other proteins (Gerst, 1999) with unclear roles. However UNC-18, UNC-13, and synaptotagmin are the best-characterized SNARE-interacting proteins.

UNC-13 promotes the open configuration of UNC-64/syntaxin required for the SNARE complex formation (Dulubova et al., 1999; Richmond et al., 2001). This protein is highly enriched at the presynaptic terminals (Kohn et al., 2000). The unc-13 mutants are aldicarb-resistant (Nguyen et al., 1995) but have normal responses to the muscle ACh receptor agonist levamisole, indicating

that the neurotransmission defect is presynaptic. The evoked responses are completely abolished and the endogenous synaptic events are virtually eliminated in the most severe mutant, the 13(s69) allele. Vesicles in contact with the membrane are accumulated in unc-13 mutant terminals suggesting a synaptic defect downstream of docking. Reduced hyperosmotic saline responses, a measure of the readily releasable pool of vesicles, also indicated that vesicles are fusion incompetent in unc-13 mutants (Richmond et al., 1999). UNC-10/Rim also interacts with UNC-13 (Koushika et al., 2001). A possible simplified mechanism of priming involves UNC-10 interaction with RAB-3, which signalises to UNC-13 the arrival of a synaptic vesicle, which in turn promotes the opened form of UNC-64/syntaxin and formation of SNARE complex. TOM-1, ortholog to vertebrate tomosyn, is a cytosolic syntaxin-binding protein implicated in the modulation of exocytosis in C. elegans. TOM-1 interacts with UNC-64/syntaxin and RIC-4/SNAP-25 to form tomosyn SNARE complexes predicted to be nonfusogenic, which compete with SNARE complex formation and thus inhibit vesicle priming. tom-1 mutants exhibit enhanced release time and suppress the priming defects of unc-13 mutants (Gracheva et al., 2006).

Unlike constitutive exocytosis, in which vesicle fusion occurs without an external stimulus, neurotransmitter-filled synaptic vesicles accumulate at the presynaptic terminal and undergo exocytotic membrane fusion being triggered only by the Ca²⁺ influx. The best-characterized putative Ca²⁺ sensor in triggering neurotransmitter release is the vesicle associated protein synaptotagmin (Andrews and Chakrabarti, 2005; Chapman, 2002; Geppert et al., 1994; Littleton et al., 1993). In C. elegans, snt-1 mutants (the gene encoding synaptotagmin) are severely uncoordinated and exhibit synaptic transmission defects (Nonet et al., 1993). However snt-1 appears to be also involved in vesicle endocytosis (Jorgensen et al., 1995).

Currently fusion model proposes that Ca²⁺ influx triggers the binding of synaptotagmin-1 to the SNARE complex and its penetration into the plasma membrane, leading to membrane fusion via a not yet understood mechanism. After fusion, the SNARE complex is dissociated and SNARE components are recycled for the next round of exocytosis (Li and Chin, 2003). Also, synaptic vesicle proteins are dispersed within the plasma membrane. A rapid mechanism for the recovery of SVs, called “kiss and run”, presumes that vesicles momentarily fuse with the plasma Figure 9. A) Clathrin-mediated and B) “kiss and

run” mevhanisms for the recovery of synaptic vesicles (from Richmond and Broadie, 2002).

membrane to form a fusion pore (kiss) and then undergo rapid dynamin-dependent fission (run)(Figure 9). The vesicle never collapses into the plasma membrane (Fesce and Meldolesi, 1999). The main mechanism involved in the recovery of synaptic membranes and proteins involves clathrin-mediated endocytosis, in a four-step recycling process: recruitment, budding, fission and uncoating (Figure 10).

The recruitment of adaptors for clathrin and synaptic vesicle proteins represents the first step in endocytosis of SVs. The signals that mark and trigger the recovery of the SVs membrane are unknown. One possibility is that synaptotagmin, a component of vesicle membranes which after SVs fusion resides on plasma membranes, might be the signal to recruit the endocytic complex. The snt-1 mutant exhibits a depletion of SVs at the neuromuscular junction and also the other synaptic proteins are diffusely distributed in the plasma membrane indicating that in this mutant the recycling of SVs is impaired (Jorgensen et al., 1995). Furthermore, SNT-1 binds to clathrin adaptor complex AP2, which recruits clathrin to the region marked for recycling (Zhang et al., 1994).

Synaptojanin, encoded by unc-26 in C.

elegans, is a polyphosphoinositide phosphatase implicated in the formation of the recruitment complex by cleaving phosphates at the 3-, 4- and 5- positions from polyphosphoinositides (Harris et al., 2000). SV are depleted in unc-26 mutants and the accumulation of PIP2 results in sequestration of SNT-1 domains involved in the AP binding, impeding clathrin recruitment (Schiavo et al., 1996). PIP2 binds also to AP2, blocking clathrin binding-site. Synaptic vesicle proteins must be also recruited to the sites of clathrin assembly. Some SV proteins need specialized adaptors. For example, the clathrin assembly protein AP180, encoded by unc-11 in C. elegans, recruits synaptobrevin-1. In unc-11 mutants, SNB-1 is diffusely distributed in the

Figure 10. Clathrin-mediated endocytosis has four different stages: recruitment, budding, fission and uncoating (from Harris et al., 2001).

plasma membrane indicating that is not recovered from the membrane. In these mutants, endocytosis still occurs, although the vesicle diameters are increased (Nonet et al., 1999).

In the second step of synaptic vesicle recycling, assembly of the clathrin matrix pulls the membrane into the cell to form a budding vesicle. The size of recycled SV is regulated by specific factors. As described above, AP180 clathrin adaptor protein plays a role in the regulation of SV size. In unc-11 mutants the synaptic vesicles reach up to 40 nm in diameter, compared with normal SV, which have 30nm (Nonet et al., 1999). The fact that endocytosis still occurs in unc-11 mutants suggests that AP180 is not essential in clathrin recruitment, instead might regulates the angle between the clathrin triskelions at the vertices (Harris et al., 2001).

During the fission step of SV recycling, the budded vesicle is cleaved from the plasma membrane. This separation requires dynamin, a small GTPase that forms a collar around the neck of the budding vesicle. The collar is constricted upon GTP hydrolysis, thereby releasing the vesicle from the plasma membrane (Sweitzer and Hinshaw, 1998). These endocytic intermediate structures are rarely observed in normal synapses, but can be observed when endocytosis is blocked prior to fission. Temperature-sensitive mutations in the C. elegans dynamin gene, dyn-1 result in a lethal phenotype when grown at the restrictive temperature of 25°C. Moreover, animals grown at the permissive temperature but subsequently placed at trestrictive temperature for 20 min become uncoordinated and have pharyngeal pumping, defecation, and egg-laying defects. These data suggest one of two possibilities: the dynamin protein made in dyn-1(ky51) mutants is functional at low temperatures but defective at high temperatures, or alternatively endocytosis in C. elegans is a temperature-sensitive process (Clark et al., 1997). Dynamin interacts with several proteins, including Eps15 and endophilin.

Eps15, an EH-domain containing protein was implicated in several vesicle-sorting pathways, including endocytosis. The 1 gene encodes the C. elegans Eps15 homolog. ehs-1 mutants exhibit locomotory defects, aldicarb resistance and vesicle depletion at raised temperatures. Most dyn-1;ehs-1 double-mutant animals are dead at 18 °C, which is normally a permissive temperature for both the dyn-1 and ehs-1 mutants. The few surviving animals are severely uncoordinated and slow growing (Salcini et al., 2001). Together, these results suggest that Eps15 has a non-essential accessory role in endocytosis; in particular, Eps15 may stabilize endocytosis at high temperatures, which might be important because the lipid fluidity might be significantly altered at high temperatures.

Endophilin, an SH3 domain-containing protein interacts with proline rich domains of both dynamin and synaptojanin (Ringstad et al., 1997). C. elegans endophilin (UNC-57) localizes to synapses. The unc-57 mutants are uncoordinated and at the ultrastructural level contain arrested coated and uncoated invaginated pits leading to 70% less synaptic vesicles than wild-type (Schuske et al., 2003). Endophilin might be an adaptor protein involved in the recruitment of synaptojanin (UNC-26) to the sites of endocytosis. Precisely how the loss of

these functional partners affects the fission is unclear; either the lipid shaping function of endophilin or the degradation of PIP2 by synaptojanin may regulate the fission step (Harris et al., 2000; Schuske et al., 2003).

During uncoating, the final step of SV recycling that occurs after the vesicle has been released from the plasma membrane, both the clathrin cage and adaptor proteins disengage from the vesicle. In vitro, the molecular chaperone Hsc70 uncoats the clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin (Rothman and Schmid, 1986; Ungewickell et al., 1997). The worms in which auxilin has been disrupted by RNAi, are unviable and display an accumulation of membrane-associated clathrin. This is consistent with clathrin heavy chain accumulating on vesicles in the auxilin-defective animals.

Overexpression of the clathrin::GFP protein in auxilin RNAi-treated worms allows the worms to grow into adults, suggesting that in the auxilin-defective animals, the amount of clathrin is limited (Greener et al., 2001).

After uncoating, vesicles may directly re-enter the vesicle pool or fuse with early endosomes for protein sorting and vesicle budding into new synaptic vesicles, thereby completing the cycle.