Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

Abstract

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion

“…Here, we present additional data that may help us understand the influence that cpx1 may have on the vesicle recycling pathway. As we have previously described ( Khounlo et al, 2021 ), we are able to make qualitative inferences about the diameter of the pore from the intensity of the fluorescence time trace. As the fusion pore dilates, the vesicle might deform, which leads to a sharp increase in florescence seen on the time trace ( Figure 2B ).…”

“…However, as the fusion pore forms and expands, we expect the shape of the vesicle to deform in such a way that the dye inside the vesicle is brought closer to the surface and deeper into the evanescent layer. This would result in an increase in fluorescence intensity ( Khounlo et al, 2021 ). Once the fusion pore reaches a critical diameter (∼6 nm), it then allows the release of the dyes from the vesicle into the gap between the bilayer and the quartz surface.…”

“…Khounlo and coworkers, using the same technique, probed the internal content release and large pore formation in SNARE-only case ( Khounlo et al, 2021 ). It was found that only approximately ∼38% of the docked vesicles advanced to the large pore stage while ∼62% demonstrated small or no pore formation, which results in no internal content release.…”

“…As described previously ( Khounlo et al, 2021 ), a quartz slide along with a cover slip is hydroxylated by boiling in piranha solution composed of a 1:1 mixture of sulfuric acid and 30% hydrogen peroxide for 15 min. The cover slip and slide are rinsed in ddH2O 3 times to remove the piranha solution.…”

“…To investigate cpx1’s role in fusion pore expansion and ultimately, its role in vesicle recycling, we employ an in vitro single vesicle-to-supported bilayer fusion assay ( Liu et al, 2005 ; Khounlo et al, 2021 ). This assay allows us to monitor, in real time, cpx1’s effect on SNARE mediated fusion pore expansion.…”