Function of Drosophila Synaptotagmins in membrane trafficking at synapses

Abstract

The Synaptotagmin (SYT) family of proteins play key roles in regulating membrane trafficking at neuronal synapses. Using both Ca²⁺-dependent and Ca²⁺-independent interactions, several SYT isoforms participate in synchronous and asynchronous fusion of synaptic vesicles (SVs) while preventing spontaneous release that occurs in the absence of stimulation. Changes in the function or abundance of the SYT1 and SYT7 isoforms alter the number and route by which SVs fuse at nerve terminals. Several SYT family members also regulate trafficking of other subcellular organelles at synapses, including dense core vesicles (DCV), exosomes, and postsynaptic vesicles. Although SYTs are linked to trafficking of multiple classes of synaptic membrane compartments, how and when they interact with lipids, the SNARE machinery and other release effectors are still being elucidated. Given mutations in the SYT family cause disorders in both the central and peripheral nervous system in humans, ongoing efforts are defining how these proteins regulate vesicle trafficking within distinct neuronal compartments. Here, we review the Drosophila SYT family and examine their role in synaptic communication. Studies in this invertebrate model have revealed key similarities and several differences with the predicted activity of their mammalian counterparts. In addition, we highlight the remaining areas of uncertainty in the field and describe outstanding questions on how the SYT family regulates membrane trafficking at nerve terminals.

Keywords: Drosophila; Exocytosis; Neurotransmitter release; Synapse; Synaptic vesicle; Synaptotagmin.

Fig. 1

Regulation of synchronous and asynchronous SV fusion. a Model depicting phases of synchronous and asynchronous release after nerve stimulation (arrow). b Structure of the fusion machinery that bridges the SV and plasma membrane, with SYT1 (grey) and the SNARE complex (Syntaxin—red; SNAP25—green; Synaptobrevin—blue). c Model of synchronous and asynchronous release in relation to presynaptic Ca²⁺ entry (shaded). Following Ca²⁺ influx, SVs fuse during a synchronous phase at AZs that occurs within milliseconds. SYT1 acts as the Ca²⁺ sensor for synchronous release and resides on SVs. A slower asynchronous component can last for hundreds of milliseconds and include fusion of SVs farther from release sites. SYT7 has emerged as a candidate for the asynchronous Ca²⁺ sensor with higher affinity than SYT1 for Ca²⁺ binding. The role of SYT7 is still controversial, with studies in Drosophila suggesting it controls SV availability and fusogenicity

Fig. 2

The Drosophila larval NMJ as a model for synaptic function. a Immunolabeling of the nerve terminal showing AZs (anti-BRP, magenta) and post-synaptic densities (anti-Glutamate Receptor III staining, green) at a larval NMJ. b EM of a single synaptic bouton with an AZ T-bar denoted (arrow). c The left panel shows a model of synaptic boutons with multiple individual AZs (left). The middle panel shows an NMJ expressing the presynaptic Ca²⁺ channel (Cac-GFP, green) and evoked SV release events (red) visualized with a modified jRGECO Ca²⁺ indicator expressed postsynaptically. The right panel shows quantal imaging of evoked release probability at the same NMJ, revealing heterogeneity in AZ strength as noted on the color-coded heat map. a Modified from [105], b modified from [82], and c modified from [22]

Fig. 3

Conservation, abundance and localization of Drosophila SYTs. a Phylogenetic tree of SYT homologs in Drosophila melanogaster (d), Mus musculus (m), and Homo sapiens (h). The SYT1, SYT4, SYT7, SYT12, and SYT14 subfamilies are highlighted. The sequences of each SYT were extracted from NCBI and the tree was generated using neighbor clustering algorithm. b Expression level of Drosophila Syt genes in larval and adult brain using RNAseq. c Expression of endogenously CRISPR-tagged SYT7-GFP compared to the AZ protein BRP in a single larval NMJ bouton. SYT7 surrounds AZs and localizes to an interconnected tubular membrane compartment within the peri-AZ. d Localization of SYT1 to SVs and SYT4 to postsynaptic puncta at the larval NMJ using immunocytochemistry. The motor axon is stained with anti-HRP (green). e Model of the subcellular localization of Drosophila SYTs. SYT1 is attached to SVs and triggers synchronous SV fusion. SYT7 localizes to an internal peri-AZ compartment and negatively regulates SV re-entry into the readily releasable pool and SV fusogenicity. SYT4 localizes to presynaptic exosomes that are released from multi-vesicular bodies (MVBs) to transfer the protein to the postsynaptic compartment where it mediates retrograde signaling. The graph in panel b was generated by plotting gene expression levels in the CNS of 7-day-old males reported in [368], panel c was modified from [100], and panel d was modified from [82]

Fig. 4

Function of SYT1 in SV fusion. a Syt1 null mutants reduce synchronous fusion and enhance asynchronous release and mini frequency. Rescue with a Syt1 transgene with defective Ca²⁺ binding to C2A(*) and C2B(*) fails to support synchronous fusion and causes higher rates of spontaneous release. b Overexpression of a C2B Ca²⁺ binding mutant (D1,2N) suppresses release compared to overexpression of wildtype SYT1. Twenty essential residues mapping to C2A (blue) or C2B (magenta) were identified in an intragenic suppressor screen that blocked the dominant-negative effects. c. Structure of the primary and tripartite interface of the SYT1/SNARE/CPX complex. d Location of mutations disrupting the primary SNARE interface on the SYT1 C2B domain. The polybasic stretch is shown in grey and localizes to the opposite C2B surface. SNARE-binding mutations fail to rescue release defects in Syt1 null mutants (right panel). e Location of the R250H mutation at the SYT1 dimer interface that disrupts oligomerization. This mutation reduces SV release (right panel), though not as severely as SNARE-binding mutants. a Modified from [49], b, d, and e modified from [62], and c modified from [38]

Fig. 5

Enhanced release and SV replenishment in Syt7 mutants. a Western analysis of Drosophila brain extracts separated on a 10–30% sucrose gradient. SYX1 labels the plasma membrane (left-most fraction) with SVs (n-SYB/SYT1) in intermediate fractions. SYT7 and SYT4 fractionate with other internal membrane compartments to distinct regions of the gradient. b Evoked release is increased in Syt7 null mutants (Syt7^M1) and Syt7 heterozygotes. c Optical mapping of release probability at larval NMJs demonstrate Syt7 mutant AZs are shifted to higher P_r compared to controls. d Syt7 mutants undergo rapid depression during a stimulation train and recover their releasable SV pool more quickly than controls. Heterozygotes show an intermediate phenotype. Panel a modified from [100] and panels b–d modified from [82]

Fig. 6

SYT4 controls retrograde signaling at Drosophila NMJs. a Drosophila NMJs display a robust form of activity-dependent presynaptic plasticity mediated by increased spontaneous release after high frequency stimulation (HFS). Optical imaging of spontaneous SV releases rates are shown before and after stimulation. Note the different y-axis scale on the post-stimulation map. b The enhanced spontaneous release observed at control NMJs after stimulation is abolished in Syt4 mutants. Arrows denote the timing of HFS to the motor axon. c Screen for muscle RNAi knockdown of loci that disrupt postsynaptic membrane expression of a SYT4-pHlourin (SYT4-pH) construct. Control SYT4-pH localization to the postsynaptic membrane is shown on the left. Syx4‐RNAi reduces membrane SYT4-pH and causes a redistribution of the protein to cytoplasmic puncta within the muscle (right). The motoneuron is stained with anti-HRP (red). a Modified from [95], b modified from [104], and c modified from [105]