Rate and extent of polyethylene glycol-induced large vesicle fusion monitored by bilayer and internal contents mixing

Abstract

Poly(ethylene glycol) (PEG) of average molecular weight 8000 was used to mediate the fusion of large unilamellar vesicles composed of dipalmitoylphosphatidylcholine. Fusion was monitored by fluorescence assays of lipid mixing and aqueous contents mixing. The extent of lipid mixing, as monitored by DPHpPC fluorescence lifetime, indicated that large unilamellar vesicles underwent a single fusion cycle when incubated with PEG and subsequently diluted into buffer. The ANTS/DPX assays for contents mixing and leakage indicated that, while addition and dilution of PEG were accompanied by extensive contents leakage, this occurred on a much different time scale as compared to contents mixing. Both the lipid-mixing and contents-mixing assays gave comparable estimates for the number of rounds of fusion that occurred in a given time following PEG addition, although the contents-mixing assay always yielded an estimate 10-15% larger than the lipid-mixing assay. These assays were used to evaluate several factors purported to influence PEG-induced fusion. First, the initial rate of fusion was found to be dependent on PEG concentration in the range of 0-35 wt %, while the extent of fusion was not. In addition, a substantial rate enhancement occurred when vesicles were incubated with greater than 26% PEG. Second, the creation of an osmotic gradient upon dilution of vesicle-PEG mixtures was shown to have no effect on either the extent or the initial rate of fusion. Consistent with this observation, both contents and lipid mixing were found to occur prior to and independent of the dilution of the PEG-vesicle suspension. Third, impurities, either present in our commercially available PEG or added to vesicle-PEG mixtures, also had no effect on the rate or extent of fusion. Fourth, another dehydrating polymer, dextran (average mol wt 9000), was capable of promoting fusion, though at a much lower rate than PEG. These results suggest that even partial bilayer dehydration accompanied by vesicle collapse and close interbilayer contact may be sufficient to induce vesicle fusion.

Surface dielectric constant, surface hydrophobicity and membrane fusion

“…In the presence of 30 wt% of PEG, where fusion of either PC or PS vesicles was observed [13,22], the surface dielectric constant decreased to about 12 for the PS membrane which is approximately the same value as those for cation-induced fusion. However, for the PC membrane it decreased to only 16, indicating that the membrane surfaces are still more hydrophilic than the PS membrane surface when fusion occurs.…”

“…The withdrawal of free water from the vesicles by PEG could cause them to flatten. Thus, two lipid vesicles might fuse due to their stress on the highly curved boundaries as proposed by MacDonald [13], and Parente and Lentz [22]. The detergent-like properties of PEG are another possibility of the bilayer destabilization [2].…”

Diffusive Dynamics of Vesicles Tethered to a Fluid Supported Bilayer by Single-Particle Tracking

“…PEG has been shown to enhance vesicle-vesicle interactions and even cause fusion for certain lipid compositions in bulk suspensions of vesicles. [21] Immediately upon addition of buffer containing PEG 8000 at concentrations that do not cause irreversible aggregation or fusion between bulk and tethered vesicles, tethered vesicles were observed to stop moving. This effect can be visualized by using a 2-lane microfluidic device where 5% w/v PEG 8000-containing buffer is flowed over one half tethered vesicles (100 nm, 1% TR-DHPE) of the microscope field of view, and PEG-free PBS buffer is flowed over the other half (see Movie 2, supporting information).…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…Membrane fusion reactions have been reconstituted from purified components in vitro, including Ca 2ϩ -and polyethylene glycol-stimulated fusion (10,11), viral fusion (12), and SNAREmediated fusion (13). Regulation of SNARE-dependent membrane fusion by the SNARE-interacting protein Sec1/Munc18 (SM) (14,15) and the SNARE-binding proteins synaptotagmin and complexin (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) has also been reconstituted.…”