Formation of phase separated vesicles by double layer cDICE

Dürre, Katharina; Bausch, Andreas R.

doi:10.1039/c8sm02491j

Cited by 20 publications

(16 citation statements)

References 32 publications

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“…In the original cDICE paper, 28 as well as in other follow-up studies, 29 , 30 , 36 , 37 injection capillaries were pulled from glass tubes to final orifice diameters of a maximum of 20 μm. Since we found these narrow glass capillaries to be a significant source of experimental variation and problems due to easy clogging of the orifice, we instead used commercially available fused silica capillary tubing with larger diameters (25, 50, and 100 μm) to allow for more consistent results, as previously used by Litschel et al ( 33 ) We found that using all three capillary sizes, our chloroform-based lipid-in-oil dispersion and optimized workflow led to high yields of GUVs with a mean diameter of 12 μm and coefficient of variation of 47% for a capillary size of 100 μm and rotation speed of 1900 rpm ( Figure 1 d).…”

Section: Resultsmentioning

confidence: 99%

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

et al. 2021

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Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

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Section: Resultsmentioning

confidence: 99%

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

et al. 2021

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“…To ensure that the lipid composition of vesicles did not vary with their size, we made careful choices about how we produced vesicles. Because different techniques incorporate different ratios of lipids into vesicles (25)(26)(27), we produced all vesicles by the same technique: electroformation (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

Direct imaging of liquid domains in membranes by cryo-electron tomography

Cornell

Mileant

Thakkar

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

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“…While the droplet crosses the interfaces of oil and water, it is step-wise coated with both leaflets of a lipid bilayer, and emerges as a unilamellar vesicle of the size of the initial droplet. [357] In 2019, Durre and Bausch [358] described a loading variant of cDICE to introduce cholesterol into lipid bilayers, enabling reliable formation of phase-separated vesicles. Another interesting variant was developed by Morita et al [359] It produces cell-sized liposomes of well-controlled lipid composition by means of droplet-shooting and size-filtration in a centrifuge.…”

Section: Microfluidics-based Techniques To Fabricate Protocellsmentioning

confidence: 99%

Protocells: Milestones and Recent Advances

Gözen

Köksal

Põldsalu

et al. 2022

Small

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to assemble astonishing pieces of a complicated puzzle, and realized that further advancement requires input from multiple branches of science, not just biology as the primary life science. Detailed hypotheses have been established about the different scenarios of the emergence of life, including the "RNA world," [1] the "lipid world," [2] "replicator first," [3] "metabolism first," [4][5][6] and others. Although the origin of life is still surrounded by many open questions, our understanding of chemical, physicochemical, and biochemical processes possibly involved in the ancient events preceding Darwinian evolution has seen much progress. We have come a long way from Leduc's physicochemical, inorganic matter-centered view on the beginning of the evolution, yet the matter of the transition from nonliving to living matter still remains largely unsolved, and one of the great scientific problems of our time.The phylogenetic tree of different living domains reflects that life has evolved from simple to more complex structures, i.e., from single-to multicellular organisms. The oldest fossil evidence dating back 3.5 Gy (billion years) comes from stromatolites, [7][8][9][10] microorganismal residues in sedimentary rocks. [11] There appears to be a gap of knowledge regarding the period of evolution between the first primitive hypothetical cells and the fossilized ancient bacteria, which can be considered as an already advanced form of life. [12] It is highly likely that intermediate primitive cell precursors preceded the single-cell organisms. The hypothetical prebiotic structures that were the stepping stone to first self-sustaining living cells are commonly termed "protocells." The possibility of a strong link between the formation of protocells and the origin of life can today be reasonably assumed.One cannot easily proceed in the context of the evolution of cell-based organisms without briefly illuminating the concept of life as we know it on our planet. Over time, different requirements have been proposed for an entity to be considered alive. According to Tibor Ganti's chemoton model, [13] a protocell contains three autocatalytic subsystems: a membrane subsystem that keeps the components together and intact, a metabolic subsystem that captures energy and material resources, and an information subsystem that processes and transfers heritable information to progeny. To be considered alive, these subsystems must be unified and function co-operatively for the survival and evolution of the supersystem. Pohorille and Deamer suggested a modified set of 7 criteria related to the chemoton. [14] At about the same time, Oro defined the requirements by 10 characteristic features. [15] Despite their differences, these descriptions align well with NASA's broader definition of life: "a self-sustaining chemical system capable of Darwinian evolution."The origin of life is still one of humankind's great mysteries. At the transition between nonliving and living matter, protocells, initially featureless aggregates of abiotic matter, ga...

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Formation of phase separated vesicles by double layer cDICE

Abstract: Here we present a modified version of cDICE that allows incorporation of cholesterol into lipid bilayers and enables the reproducible formation of phase-separated vesicles.

Cited by 20 publications

References 32 publications

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

Direct imaging of liquid domains in membranes by cryo-electron tomography

Protocells: Milestones and Recent Advances

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