Layer-by-layer cell membrane assembly

Abstract

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion of both purified and in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.Phospholipid membranes are complex supramolecular assemblies involved in every aspect of biological function. They drive Darwinian evolution by coencapsulating the cellular metabolism and genetics, and they are universally the means by which Eukarya spatially organize their diverse subcellular metabolic processes. These structures have many interrelated traits, such as curvature, fluidity, lamellarity, and composition 1 . While the role of membrane bilayers in shaping the structure and function of their associated proteins is well studied 2 , the biological significance of more subtle membrane characteristics, such as compositional asymmetry and membrane curvature , are burgeoning areas of investigation 3,4 . Elucidating their biochemical roles will require the ability to synthesize increasingly complex membranes 5 that exhibit the full scope of these traits.Systematic membrane assembly from simple starting materials is a long-standing challenge. Bulk-scale membrane assembly, such as phospholipid film hydration 6 , yields no control over vesicle size, content, membrane composition, and lamellarity. These highly heterogeneous preparations nonetheless pervade in vitro studies of membrane biochemistry because they require no specialized equipment and the experimenter can hunt for the right Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Correspondence: briandna@scripps.edu. Additional Information: The authors declare no competing financial interests.Author Contributions: B.M.P. and S.M. conceived and designed the experiments, analyzed the resulting data and co-authored the paper. S.M. executed all experimental work. [7][8][9][10] . The products of microfluidic assembly are strikingly homogeneous in size (1-5% CV), encapsu...

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