Lehrstuhl für Zellbiophysik E27, Technische Universität München, James-Franck-Straße 1, 85748 Garching, Germany.
Soft Matter. 2019 Dec 4;15(47):9676-9681. doi: 10.1039/c8sm02491j.
Recently, continuous droplet interface crossing encapsulation (cDICE) was developed, which allows fast and efficient production of giant unilamellar vesicles (GUVs) under high salt conditions, at low temperature and with low consumption of the encapsulated proteins. Unfortunately, cholesterol encapsulation within the lipid bilayer was not efficient for the cDICE protocol so far and thus the formation of phase separated vesicles was limited. 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. We show that cholesterol incorporation relies on the amount of mineral oil in the lipid-oil emulsions, which is essential for protein encapsulation inside GUVs by cDICE. The possibility of creating phase separated vesicles by cDICE will enable the study of the interdependence between phase separation and cytoskeletal proteins under confinement.
最近，连续液滴界面跨越包封（cDICE）技术得到了发展，该技术允许在高盐条件下、低温下且对包封蛋白的消耗较低的情况下快速有效地生产巨大的单层囊泡（GUVs）。不幸的是，胆固醇在脂质双层内的包封效率不高，因此相分离囊泡的形成受到限制。在这里，我们提出了 cDICE 的一个改进版本，该版本允许胆固醇掺入脂质双层，并能够重复形成相分离的囊泡。我们表明，胆固醇的掺入依赖于脂质-油乳液中的矿物油的量，这对于 cDICE 内 GUV 内蛋白质的包封是必不可少的。通过 cDICE 形成相分离囊泡的可能性将使在受限条件下研究相分离和细胞骨架蛋白之间的相互依赖关系成为可能。
