Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study

Abstract

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy-and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.

Directed Growth of Biomimetic Microcompartments

“…In some of the examples fusion has led to spontaneous budding, correlating to predictions made by theoretical models, [91] which has been put in the context of growth and division cycles for self-reproduction. [118] In contrast, when mediated by high charge density, the fusion of positively charged liposomes induces extensive area increase of the negative GUVs [89,119] (see also Figure 2D). [112] Membrane fusion of small liposomes has been used for decades [113,114] and only more recently fusion using giant vesicles has been reported.…”

“…[95] The latter observation was ascribed to the low amount of product due to the limited encapsulation, which will be difficult to overcome unless efficient transport mechanisms for the various precursors are established. [89] The change occurs within seconds. [96] Although the authors did not specifically follow the vesicle growth, they identified practical barriers causing the low lipid synthesis rate, speculated about transport mechanisms for acyl-CoA, and discussed relevant crowding and confinement effects.…”

“…Recently, this cell-free approach was extended to a more comprehensive pathway, starting from acyl-CoA and glycerol-3-phosphate, and the liposome compartments were equipped with α-hemolysin to facilitate uptake of small polar molecules. [89] Copyright 2019, Elsevier. Another example in a similar direction was the biochemical synthesis of palmitate based on a FAS type I enzyme, encapsulated in liposomes, [97] where vesicle growth has indeed been observed C) Electrofusion relies on poration of two opposing membranes exposed to strong electric fields.…”

Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review

“…In another work, Shiomi et al showed that electrofusion can be achieved in a closed microfluidic device but the setup also required the use external optical traps for vesicle selection/positioning. [88] In this work, microfluidic valves allow rapid and real-time monitoring of the fusion events (see Figure 3e). [67,87,92] Positioning of the vesicles relative to the electrodes is achieved either by alignment using the electric field itself, [67] growing them on the electrodes, [92] or trapping in PDMS posts.…”

“…[85] They also showed that GUVs containing a simple set of cytoskeletal proteins Adv. [88] Copyright 2019, Elsevier. 2019, 3, 1800318 [36] Copyright 2014, American Chemical Society.…”