Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores

Abstract

Here we introduce ApoE-based nanolipoprotein particle (NLP)-a 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 sizes show 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 with 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.

Figure 1

Assembly and characterization of ApoE nanolipoprotein particles. (A) Schematics of the protocol to assemble ApoE NLP complexes. (B) Elution profiles for NLPs of different ApoE/lipid ratio purified using size exclusion chromatography. The ND and 1:45, 1:120, 1:180 NLPs were isolated on Superose 6 column and 1:290 NLPs on Sephacryl S-500 column. The relative elution volume for the two columns were calculated using the void volume of 8 ml and 40 ml for Superose 6 and Sephacryl S-500 columns respectively. (C) Negative stain electron microscopy analysis of the ApoE NLPs assembled with different ApoE/lipid ratios. Representative micrographs (left) and the size distribution estimated using a minimum of 200 individual particles using Image J software (right) are shown.

Figure 2

VAMP2 containing NLPs (vNLPs) as fusion partners. (A) Elution profiles of empty and VAMP2 containing NLPs on Superose 6 SEC column. Inset: Coomassie stain SDS-PAGE analysis of the elution peaks. (B) Lipid mixing monitored by dequenching of NBD fluorescence following the fusion of vNLP containing NBD and Rhodamine to acceptor t-SUVs. (C) Formation of the fusion pore monitored by the efflux of calcium entrapped in the t-SUVs by inclusion of calcium-sensitive fluorophore, Mag-Fluo-4 in the external medium. In both cases, Nanodiscs containing same number (~9 copies) of VAMP2 (red curve) is shown for comparison and control experiments with SNARE-free NLPs (grey) or addition of cytoplasmic domain of VAMP2 (CDV, green) are also included (D) Some NBD fluorescence included in the NLPs was protected from dithionite added externally after-fusion for vNLPs (red) confirming that they undergo full fusion and that the fusion pore reseal effectively. In contrast, it is completely quenched with SNARE-free NLP (black).

Figure 3

NLPs accommodate the dilation of the fusion pore. (A) Schematics of the dextran release assay. Amount of encapsulated dextrans 3000 MW (stokes radius ~1 nm), 10,000 MW (~2.3 nm) and 40,000 MW (~4.4 nm) released in to the supernatant after fusion with vNLPs was estimated following pelleting of the vesicles. The dextran released (%) was corrected for inefficient pelleting and for non-specific leakiness/lysis. (B) SDS-PAGE analysis confirms that equal amounts of t-SNAREs have been incorporated in each dextran containing t-SUVs. The SNARE-free and VAMP2 (9 copies) containing ND and NLP used in this analysis are also shown (C) Normalized dextran released in the medium shows that dextrans of all sizes are efficiently released with NLPs as compared to the NDs confirming that NLPs accommodate expansion of the nucleated fusion pore up to at least ~9 nm

Figure 4

Effect of VAMP2 copy number on fusion pore dynamics. (A) NLPs with desired number of VAMP2 copy was generated by adjusting the ApoE/VAMP2 input ratio. Based on the coomassie stained SDS-PAGE analysis, the number of copies of VAMP2 per NLP was estimated by densitometry assuming each NLP is formed by 6 copies of ApoE protein. The size and particle distribution of vNLPs was determined by negative stain EM analysis. (B) All vNLPs tested drove lipid mixing as monitored by NBD dequenching assay, with rate and extent of fusion correlated to the copy number. (C) Calcium release assays of vNLP with different VAMP2 copies shows efficient efflux starting with NLP containing at least 3–4 copies of VAMP2 (vNLP4), with little or no calcium efflux was detectable with lower copy number (vNLP1, orange curve). (D) Dextran release assay shows that increasing the number of copies of VAMP2 per NLP enhances the efflux of dextran, including the larger (10,000 MW and 40,000 MW) dextrans indicating a cooperative role for SNAREs in dilating the fusion pore.