Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

Abstract

Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca 2+ -dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1's ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.synaptotagmin | Drosophila | synaptic vesicle | neurotransmitter release | exocytosis N eurotransmitter release requires temporal and spatial coupling of action potential-triggered Ca 2+ influx to synaptic vesicle fusion (1). The core fusion machine contains SNARE proteins found on the synaptic vesicle (v-SNAREs) and plasma membrane (t-SNAREs) that assemble into a four-helix bundle to bring the two bilayers into close apposition (2, 3). Besides SNAREs, Ca 2+ -binding proteins act to trigger release through fast synchronous and slow asynchronous pathways. Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that binds Ca 2+ and triggers synchronous vesicle fusion (4-9). Syt1 contains an intravesicular N-terminal tail, a single transmembrane segment, and a ∼60-residue linker that connects to two cytoplasmic Ca 2+ -binding C2 domains (10-13). Numerous Syt1 studies have focused on its cytoplasmic C2 domains, which interact with phospholipids and the SNARE complex in a Ca 2+ -dependent manner and are proposed to be the essential domains that trigger fusion (12, 14-21). In contrast, the significance of other structural elements of Syt1 remains poorly understood. Syt1 is predicted to facilitate synaptic vesicle fusion through a trans interaction with plasma membrane lipids (22-27). Tethering of Syt1 to synaptic vesicles through its transmembrane domain has been postulated to position the protein to properly target lipids and SNAREs, or to be required to generate force for pulling th...

Models of synaptotagmin‐1 to trigger Ca2+‐dependent vesicle fusion

“…1) In vitro fusion data might vary depending on whether full‐length Syt1 or the C2AB domain is used for fusion assay. The C2AB domain fails to rescue the phenotype of Syt1 knockout and only full‐length Syt1, not the C2AB domain, efficiently reproduces Ca 2+ ‐dependent fusion in the in vitro reconstitution system . High concentration of the C2AB domain causes clustering and aggregation of liposomes and affect fusion independently of Syt1 activity.…”

Transmission, Development, and Plasticity of Synapses

“…Syts are a family of vesicular Ca 2+ sensors with two cytoplasmic Ca 2+ binding C2 domains, termed C2A and C2B (Adolfsen and Littleton 2001;Sudhof 2004). Syt1 is one of the most well-studied vesicle trafficking proteins and functions to sense Ca 2+ influx and regulate fusion of SVs (Littleton et al 1993(Littleton et al , 2001aBroadie et al 1994;Diantonio and Schwarz 1994;Reist et al 1998;Yoshihara and Littleton 2002;Mackler et al 2002;Saraswati et al 2007;Yoshihara et al 2010;Jorquera et al 2012;Striegel et al 2012;Lee et al 2013b;Lee and Littleton 2015). Neurotransmitter release is characterized by a synchronous phase of SV fusion that occurs within milliseconds, and a slower asynchronous component that can last for hundreds of milliseconds depending on the synapse (Kaeser and Regehr 2014).…”

“…Neurotransmitter release is characterized by a synchronous phase of SV fusion that occurs within milliseconds, and a slower asynchronous component that can last for hundreds of milliseconds depending on the synapse (Kaeser and Regehr 2014). Drosophila syt1 null mutants lack the fast synchronous component of evoked fusion and show enhanced asynchronous and spontaneous release as well ( Figure 2B) (Littleton et al 1993;Yoshihara and Littleton 2002;Yoshihara et al 2010;Jorquera et al 2012;Lee et al 2013b;Lee and Littleton 2015). These data suggest that two kinetically distinct Ca 2+ sensors exist, with Syt1 mediating the rapid, synchronous component of transmitter release (Yoshihara and Littleton 2002), and a second unknown Ca 2+ sensor underlying asynchronous fusion.…”

“…Recent data indicate the subcellular localization of Syt1 may also differentially regulate these two mechanisms for SV fusion. Syt1 requires membrane tethering to SVs to activate synchronous SV release, although asynchronous release can be promoted by cytosolic versions of Syt1 lacking the transmembrane tether (Lee and Littleton 2015). To examine how Syt1 controls SV fusion, the field has generated and performed electrophysiological analysis of transgenically expressed Syt1 transgenes mutated at Ca 2+ binding sites in C2A or C2B in the background of Drosophila syt1 null mutants ( Figure 2B).…”

The first one hundred nanometers inside the pre-synaptic terminal where calcium diffusion triggers vesicular release

“…1). Such sites are Ca 2+binding proteins, located on synaptotagmins, that are involved in triggering directly or not vesicular fusion [18]. They are located precisely in this nanometric region below vesicles.…”