Solution single-vesicle assay reveals PIP₂-mediated sequential actions of synaptotagmin-1 on SNAREs

Abstract

Synaptotagmin‐1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t‐) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single‐molecule technique, alternating‐laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t‐SNARE/phosphatidylinositol 4,5‐biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin‐2 binding to t‐SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t‐SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

“…A Ca 2+ -independent role attributed to synaptotagmin was illustrated by SytI mutant, which lacks Ca 2+ binding in both its C2 domains and facilitates vesicle docking. 52 Ca 2+ -independent role of SytI is also supported by its ability to facilitate close membrane apposition in vitro even in the absence of calcium binding [24][25][26] and the recent identification of Ca 2+ -independent SNARE-binding motifs. 53 Syt XI may similarly facilitate lysosome docking to the cell membrane.…”

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

“…3E. If the lipid-mixing step were inhibited, the docked population must have accumulated in the solution as much as the reduced subpopulation of lipid-mixed vesicles (38). The fact that the docked population did not increase implies that large α-Syn oligomers inhibited the docking step, i.e., the initial complex assembly between t-SNARE and synaptobrevin-2.…”

“…Furthermore, a bulk lipid-mixing assay cannot distinguish between effects caused by differences in docking, hemifusion, or complete fusion. Our single-vesicle alternating-laser excitation (ALEX) lipid-mixing method (discussed below) can distinguish between docking and hemifusion/fusion (38), although it still cannot distinguish between hemifusion and complete fusion (35)(36)(37).…”

“…To verify this scenario, we used ALEX, a single-vesicle assay that can discriminate between docking and lipid-mixing steps (Fig. 3) (38). ALEX observes one vesicle at a time, diffusing in solution (Fig.…”

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…125 Likewise, the Ca 2+ -sensing protein, synaptotagmin, mediates neurotransmitter release via interacting with PI(4,5)P 2 and t-SNARE at the pre-synaptic axon terminal of neurons. 126 Similarly, assembly of factors required for early endosome fusion involves the recruitment of early endosome antigen 1 protein to phosphatidylinositol 3-phosphate (PI(3)P) located at the cytosolic leaflet of endosome via its FYVE domain. 127 As the availability of certain phospholipids is key in orchestrating the cellular localization of various phospholipid-binding proteins, a number of enzymatic-and non-enzymatic-based mechanisms are present to tightly regulate this process.…”