Membrane tension-mediated growth of liposomes : A step closer to synthetic cells

Abstract: Living cells are highly complex, making it an extremely challenging task to understand how they function. A possible solution is the bottom-up assembly of non-living components and building up life-like features from scratch, i.e., using synthetic cells as a tool to understand the basic characteristics of life. One such chassis for synthetic cells are liposomes, which, like the cell membrane of living cells, are made of phospholipids. As living cells grow, lipids are incorporated into their membrane in order to cope up with the volume increase of the cell. In a similar fashion, a variety of ways are currently being investigated to achieve growth of synthetic cells. Few examples include incorporation of fatty acids from the surrounding environment, reconstituting the enzymes for fatty acid or lipid biosynthesis in the liposome, or by carrying out the synthesis of artificial membrane components through the external addition of precursor molecules. Here, we demonstrate the membrane-tension mediated growth of giant unilamellar vesicles (GUVs) by fusing sub-micrometre-sized feeder vesicles to them. We use a recently developed microfluidic technique, octanol-assisted liposome assembly (OLA), to produce cell-sized (~10 μm) GUVs on-chip. Following the density-based separation of the liposomes from the waste product (1-octanol droplets), we supply small unilamellar vesicles (SUVs, ~30 nm in diameter) which act as a lipid reserve for growth by fusing with the GUVs. The lipids molecules, being very stable in bilayer conformation, require energy to reorient themselves and undergo membrane fusion. We show that increased membrane tension of GUVs can act as a sole driver to carry out multiple fusion events and cause significant growth. By placing a mass population (>1000) of GUVs in a sufficiently hypotonic solution (delta c 3−5 mM), we build up the membrane tension (~10 mN/m) driving multiple SUV-GUV fusionevents, eventually doubling the volume of a part of the population. We probe a variety of lipid compositions, including hybrid (composed of lipids and fatty acids) GUVs and find the growth to be dependent on the lipid composition. Maximum growth is obtained when using a hybrid system, as compared to pure lipids. Our results show the possibility to use a protein-freeminimal system to induce growth in a minimalistic manner and the demonstrated highthroughput microfluidic approach may have useful implications towards realizing an autonomous entity capable of undergoing a continuous growth-division cycle.