Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

Biner, Olivier; Schick, Thomas; Müller, Yannic; Ballmoos, Christoph von

doi:10.1002/1873-3468.12233

Cited by 50 publications

(102 citation statements)

References 41 publications

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“…Although somewhat unexpected, these results are not completely surprising. Other charge-mediated membrane fusion assays have reported little or no effects of a range of salts on fusion of charged membranes (63)(64)(65). For the conditions described here, electrostatic interactions between membranes of opposite charges are so strong that the presence of these ions was not able to affect fusion at a detectable level.…”

Section: A Fret-based Single Guv Assay: Assessing the Membrane Composmentioning

confidence: 59%

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

Lira

Robinson

Dimova

et al. 2019

Biophysical Journal

118

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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.

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Section: A Fret-based Single Guv Assay: Assessing the Membrane Composmentioning

confidence: 59%

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

Lira

Robinson

Dimova

et al. 2019

Biophysical Journal

118

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“…During the preparation of this manuscript, an article was published reporting the reconstitution of bacterial membrane transport proteins using charge-mediated fusion [46]. In this study, Biner et al .…”

Section: Discussionmentioning

confidence: 99%

“…In this study, Biner et al . fused negatively-charged lipid GUV (neutral DOPC + anionic DOPG) with positively-charged proteoliposome SUV (neutral DOPC + cationic DOTAP) containing two membrane protein complexes from bacteria, with successful preservation of functional activity [46]. Here we fused positively-charged lipid GUV with negatively-charged SUV proteoliposomes using the 110 kDa Ca 2+ -transport ATPase (SERCA) from rabbit muscle as the model protein.…”

Section: Discussionmentioning

confidence: 99%

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Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Bian

Autry

Casemore

et al. 2016

Biochemical and Biophysical Research Communications

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We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca2+ transport by SERCA controls key processes in human cells, such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca2+ dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e.g., lipid components, SERCA preparation, and activity assay requirements). ATP-dependent Ca2+ transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca2+ indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca2+ transport using thapsigargin (TG), a small-molecule inhibitor of SERCA currently in clinical trials as a prostate cancer prodrug. The GUV system overcomes the problem of inhibitory Ca2+ accumulation for SERCA in native and reconstituted small unilamellar vesicles (SUV). We propose that charge-mediated fusion provides a widely-applicable method for GUV reconstitution of clinically-important membrane transport proteins. We conclude that GUV reconstitution is a technological advancement for evaluating small-molecule effectors of SERCA.

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“…[17a] Synthetic mimetics of cell membranes are,f or example,g iant unilamellar vesicles (GUVs), which can be formed from predefined phospholipid constituents. [182] Synthetic vesicles are used as model systems to investigate membrane protein functions,but they also serve as compartments for the bottom-up construction of minimal cellular systems.S uch biomimetic systems (e.g.G UVs and others) that contain functional parts or systems have the capacity to produce energy, [183] to run atranscriptional oscillator, [184] or to perform biochemical reactions and so represent small celllike bioreactors. [185] An example of reconstituting ac ellular phenomenon is based on the MinCDE protein system from E. coli,which has been studied in cell-free environments to identify the minimal requirements for maintaining its functionality.T he presence of the two purified proteins MinD and MinE, af lat mem-brane,a nd ATPw as sufficient to initiate the dynamic selforganization of the proteins (see Figure 5D)and result in the formation of surface waves.…”

Section: Reconstitution Of Minimal Synthetic Systemsmentioning

confidence: 99%

Synthetic Biology—The Synthesis of Biology

2017

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Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The "top-down" strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the "bottom-up" strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.

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Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

Cited by 50 publications

References 41 publications

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

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

Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Synthetic Biology—The Synthesis of Biology

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