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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.

SUBMITTER: Lira RB

PROVIDER: S-EPMC6342729 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Lira Rafael B RB Robinson Tom T Dimova Rumiana R Riske Karin A KA

Biophysical journal 20181201 1

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 ...[more]

PMID: 30579564

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Project description:Protein synthesis begins at free ribosomes or ribosomes attached with the endoplasmic reticulum (ER). Newly synthesized proteins are transported to the plasma membrane for secretion through conventional or unconventional pathways. In conventional protein secretion, proteins are transported from the ER lumen to Golgi lumen and through various other compartments to be secreted at the plasma membrane, while unconventional protein secretion bypasses the Golgi apparatus. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are involved in cargo vesicle trafficking and membrane fusion. The ER localized vesicle associated SNARE (v-SNARE) protein Sec22 plays a major role during anterograde and retrograde transport by promoting efficient membrane fusion and assisting in the assembly of higher order complexes by homodimer formation. Sec22 is not only confined to ER-Golgi intermediate compartments (ERGIC) but also facilitates formation of contact sites between ER and plasma membranes. Sec22 mutation is responsible for the development of atherosclerosis and symptoms in the brain in Alzheimer's disease and aging in humans. In the fruit fly Drosophila melanogaster, Sec22 is essential for photoreceptor morphogenesis, the wingless signaling pathway, and normal ER, Golgi, and endosome morphology. In the plant Arabidopsis thaliana, it is involved in development, and in the nematode Caenorhabditis elegans, it is in involved in the RNA interference (RNAi) pathway. In filamentous fungi, it affects cell wall integrity, growth, reproduction, pathogenicity, regulation of reactive oxygen species (ROS), expression of extracellular enzymes, and transcriptional regulation of many development related genes. This review provides a detailed account of Sec22 function, summarizes its domain structure, discusses its genetic redundancy with Ykt6, discusses what is known about its localization to discrete membranes, its contributions in conventional and unconventional autophagy, and a variety of other roles across different cellular systems ranging from higher to lower eukaryotes, and highlights some of the surprises that have originated from research on Sec22.

Dataset Information

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

Publications

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

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