Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores

Abstract

Here we introduce ApoE-based Nanolipoprotein particles (NLP) – soluble, discoidal bilayer mimetic of ~23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different size shows that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ~ 9 nm and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs were defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3–4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open and the SNARE proteins act cooperatively to dilate the nascent fusion pore.

Dilation of fusion pores by crowding of SNARE proteins

“…Conductance was low when ~15 nm MSP nanodiscs with eight copies of v-SNAREs (vMSP8) were used (Wu et al, 2016), but not affected when ~23 nm NLPs bearing 30 v-SNAREs were employed (vNLP30). These results are consistent with those of Bello et al (2016), who showed that progressively larger cargo could be released from t-SNARE liposomes during fusion with vNLPs as the v-SNARE copies per NLP was increased. Second, conductance of $n$ small pores in a single NLP would be additive, giving total conductance equal to $G_{po}=n \times g_{po}$, where $g_{po}$ is the mean open-pore conductance of a small pore.…”

“…We estimate that 4–5% of the docked vNLPs undergo fusion with the flipped t-SNARE cells over the course of ~20 min (Figure 2—figure supplement 1). In comparison, fusion between v-SNARE NLPs and t-SNARE liposomes yields a similar extent of lipid mixing over the same period (Bello et al, 2016).

10.7554/eLife.22964.004Figure 2.vNLPs induce lipid mixing when incubated with flipped t-SNARE cells (tCells).( a ) Schematic of the assay.

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Toward a unified picture of the exocytotic fusion pore

“…ApoE‐based scaffolds afford ~ 25 nm discs that can accommodate ~ 15 v‐ SNARE s per face . With these discs, the fusion pore can expand to ≳ 10 nm diameter with little obstruction from the scaffold ring . (B) NDs reconstituted with neuronal v‐ SNARE s are mixed with small liposomes reconstituted with complementary neuronal t‐ SNARE s. Bulk cargo release is monitored by an increase in the fluorescence of a cargo‐sensitive dye present in the bath.…”

“…This suggested a very small and/or transient pore could be induced by a single SNARE complex (which is sufficient to maintain two bilayers in close proximity [54]), but a larger/ longer-lived pore that allowed detectable calcium release required three or more complexes [36]. Bello et al [35] extended these studies by systematically varying both the size of cargo encapsulated into t-SNARE liposomes and v-SNARE copy numbers in larger,~23 nm NLPs. There was a positive correlation between v-SNARE copies per disc and the size of cargo that could be released efficiently [35].…”

A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution

“…We follow a protocol developed in the Rothman lab for making these discs, described in ref. 31 , and Stroeva and Krishnakumar [32]. …”