In addition to the regulation of SV exo
In addition to the regulation of SV exo- and ddhC by Ca2+ influx retrieval of SV membranes depends on the exocytic insertion of SV components into the presynaptic membrane: If exocytic SV fusion in neurons is abrogated by genetic inactivation of the core release machinery (e.g. knockout of Munc18 in mice (Verhage et al., 2000)), AP-triggered Ca2+ influx is unable to trigger endocytic uptake of presynaptic membranes. Similarly, when exocytosis is blocked acutely by botulinum neurotoxins that cleave SNARE proteins, no endocytosis is observed (Yamashita et al., 2005). Thus, SV exocytosis is required for SV endocytosis in addition to Ca2+ influx (Fig. 1B). How exocytic SV fusion triggers the initiation of SV endocytosis is unknown. One possibility is that surface-stranded SV proteins act as platforms for the plasma membrane recruitment of endocytic factors. For example, the v-SNARE synaptobrevin associates with the endocytic clathrin adaptor AP180 and its closely related ubiquitously expressed paralog CALM (Koo et al., 2015). However, loss of AP180 either on its own or together with CALM does not block or significantly delay endocytosis of SV proteins other than synaptobrevin (Koo et al., 2015). Similarly, knockout of the synaptotagmin-specific sorting adaptor stonin 2 causes missorting of synaptotagmin 1 to the neuronal surface, while the kinetics of SV endocytosis is even slightly accelerated (Kaempf et al., 2015, Kononenko et al., 2013). Furthermore, newly exocytosed synaptobrevin or synaptotagmin do not significantly contribute to SV endocytosis under stimulation conditions resulting in the exocytic fusion of the readily releasable pool of SVs (Fernandez-Alfonso et al., 2006, Gimber et al., 2015, Wienisch and Klingauf, 2006). Together these data argue that exocytosis of SVs is unlikely to initiate endocytosis by triggering the recruitment of clathrin-associated endocytic sorting adaptors. It is, however, possible that SV proteins trigger SV membrane retrieval by other mechanisms: For example, they might associate with or activate core components of the endocytic machinery, e.g. lipid metabolizing enzymes (see below) or fission factors such as endophilin (Voglmaier et al., 2006), dynamin (Daly and Ziff, 2002) or actin. Moreover, exocytosed SV proteins could trigger membrane shape changes that facilitate the formation of endocytic membrane invaginations within the periphery of the AZ akin to membrane deformation by wedge-like amphipathic helices or hairpin-loop transmembrane proteins (Doherty and McMahon, 2009, McMahon and Gallop, 2005).
Exocytic-endocytic coupling by membrane lipids In addition to the coupling of SV exocytosis and endocytosis by proteins it is conceivable that both processes are coupled by membrane lipids (Dittman and Ryan, 2009, Lauwers et al., 2016, Puchkov and Haucke, 2013). SV exocytosis and endocytosis both depend on phosphoinositides (PIPs), in particular on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Di Paolo and De Camilli, 2006). Evidence from biochemical experiments and more recent super-resolution imaging suggests that PI(4,5)P2 is concentrated at synapses, where it forms nanoclusters with an average diameter of about 70 nm (van den Bogaart et al., 2011). PI(4,5)P2 clusters at exocytic sites are marked by secretory vesicles (e.g. dense core vesicles in neuroendocrine cells) and syntaxin 1A, which binds to PI(4,5)P2 and its downstream product PI(3,4,5)P3 via its juxtamembrane region (Khuong et al., 2013, van den Bogaart et al., 2011). Depletion of PI(4,5)P2 by overexpression of a membrane-targeted variant of the PI(4,5)P2-specific PI-phosphatase synaptojanin or knockout of PIPKIγ, the main PI(4,5)P2-synthesizing enzyme at synapses, profoundly impairs exocytosis in neuroendocrine cells and in primary neurons (Di Paolo et al., 2004, Milosevic et al., 2005). PI(4,5)P2 synthesis by PIPKIγ is under activity-dependent control by the kinase Cdk5 and the phosphatase Ca2+/calcineurin, which promotes complex formation of PIPKIγ with talin, a protein linked to the presynaptic membrane via integrins (Di Paolo et al., 2002, Lee et al., 2005). Moreover, PIPKIγ is activated by small GTPases such as Arf6 (Krauss et al., 2003). Presynaptic PI(4,5)P2 facilitates exocytosis by regulating vesicular release probability, presumably via activation of PI(4,5)P2-binding effector proteins such as CAPS, Munc13, and/or synaptotagmin 1 (Lauwers et al., 2016, Martin, 2015, Wu et al., 2014a, Walter et al., 2017). The activity of PI(4,5)P2 in exocytosis likely is complemented by other negatively charged phospholipids such as phosphatidylserine (PS) and diacylglycerol (DAG), which bind to several proteins involved in SV priming and docking in the AZ. This is largely mediated by structured motifs, such as C1, C2 (e.g. found in Munc13), phosphotyrosine-binding (PTB) and plekstrin homology (PH) domains (Koch and Holt, 2012, Lauwers et al., 2016, Martin, 2015, Williams et al., 2009).