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Membrane Tension-Mediated Growth of Liposomes

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Membrane Tension-Mediated Growth of Liposomes Membrane tension-mediated growth of liposomes A step closer to synthetic cells Sai Sreekar Wunnava Venkata Degree project in biology, Master of science (2 years), 2018 Examensarbete i biologi 30 hp till masterexamen, 2018 Biology Education Centre, Uppsala University, and Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Building 58, Van der Maasweg 9, 2629 HZ Delft, The Netherlands Supervisors: Prof. Cees Dekker and Dr. Siddharth Deshpande External opponent: Dr. Disa Larsson Hammaröf Contents ABSTRACT .......................................................................................................................... 2 LIST OF ABBREVIATIONS ................................................................................................. 3 1 INTRODUCTION .......................................................................................................... 1 1.1 Bottom-up assembly of synthetic cells ....................................................................... 1 1.2 Liposome as the synthetic cell container ................................................................... 1 1.3 Growing synthetic cells ............................................................................................ 3 1.4 Membrane Fusion .................................................................................................... 4 1.5 Membrane tension as the driving force for fusion ...................................................... 6 1.6 Aim ......................................................................................................................... 8 2 METHODS AND MATERIALS ...................................................................................... 9 2.1 Octanol-assisted Liposome Assembly (OLA) ............................................................. 9 2.2 Casting of the microfluidic device ........................................................................... 12 2.3 Surface treatment with polyvinyl alcohol ................................................................ 13 2.4 Lipids used in different compositions ...................................................................... 13 2.5 Preparation of small unilamellar vesicles (SUVs) .................................................... 14 2.6 Experimental Setup ............................................................................................... 14 3 RESULTS .................................................................................................................... 16 3.1 Back-of-the-envelope calculations to figure out experimental parameters ................ 16 3.2 Growing pure lipid vesicles .................................................................................... 19 3.2.1 Using cylindrical (DOPC) and inverted cone-shaped (DOPE) lipids ..................... 19 3.2.2 Using cylindrical (DOPC), inverted cone-shaped (DOPE), and cone-shaped (LPC) lipids 21 3.3 Growing hybrid vesicles (DOPC and oleic acid) ...................................................... 25 3.4 No growth is observed in absence of membrane fusion ............................................ 29 4 DISCUSSION ............................................................................................................... 31 5 REFERENCES ............................................................................................................. 34 6 APPENDIX .................................................................................................................. 40 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 fusion events, 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-free minimal system to induce growth in a minimalistic manner and the demonstrated high- throughput microfluidic approach may have useful implications towards realizing an autonomous entity capable of undergoing a continuous growth-division cycle. Keywords: synthetic cells, liposomes, growth, membrane fusion, bottom-up biology, microfluidics LIST OF ABBREVIATIONS RNA ribonucleic acid DNA deoxyribonucleic acid DPPC dipalmitoyl phosphatidylcholine DOPC dioleoyl phosphatidylcholine SNAREs Soluble N-ethylmaleimide-sensitive factor Attachment Protein receptor PEG Polyethylene glycol SUV small unilamellar vesicles GUV giant unilamellar vesicles OLA Octanol-assisted liposome assembly OA Outer aqueous IA Inner aqueous LO lipid-carrying organic PDMS polydimethylsiloxane PVA polyvinyl alcohol DOPE dioleoyl phosphatidyl ethanolamine LPC lyso phosphatidyl choline (oleoyl-hydroxy phosphatidyl choline) PE-CF dioleoyl phosphoethanolamine-N-(carboxyfluorescein) Rh-PE dioleoyl phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) 1 INTRODUCTION 1.1 Bottom-up assembly of synthetic cells Life, as we know, is highly complex. Even one of the smallest and the simplest organisms, Mycoplasma genitalium, which has 525 known genes(Razin, 1997), is complex enough to not fully understand the functioning of these genes and the emergent properties arising from their interactions. To understand this complexity of life, one approach is to strip down the genome to just leave only the genes essential for survival. Using this top-down approach, the Mycoplasma genome has been reconstructed to make a new cell, JCVI-syn3.0, with 438 essential protein-coding genes and 35 RNA coding genes in a 531 kbp genome(Hutchison et al., 2016). Even with such minimal genome, for about 32% of the “essential” genes, the function remains unknown. An alternative way to go is a bottom-up reconstitution of a protein-based self-replicating system, from biological components, such as a reduced genome encoding for DNA, RNA and protein synthesis and encapsulating it in a membrane to form a synthetic minimal cell(Forster and Church, 2006; Jewett and Forster, 2010). Both these ways towards synthesizing life have major disadvantages of either the lack of understanding of the functions or the practical hindrances to integrating the biological subsystems which require radically different conditions for their activity. To circumvent this, one can either start assembling life completely bottom- up from simple chemical entities(Luisi, Ferri and Stano, 2006) or understand the origins of life and try to retrace the path nature took in evolving these systems to the current form(Schrum, Zhu and Szostak, 2010). However, except for the top-down approach, the bottom-up approaches would require a container/ compartment which separates the system from the environment. To this aid, most utilized container so far is a liposome, due to its resemblance with natural living cells. 1.2 Liposome as the synthetic cell container Liposome, just like the cell membrane, is composed of phospholipids assembled in a bilayer enclosing an aqueous interior. Phospholipids are amphipathic molecules with a polar head group and a hydrophobic tail, and in aqueous solutions, in order to minimize the interaction of hydrophobic tails with water, they spontaneously self-assemble to form assemblies with the head groups facing water and tails pointing away in micelles or bilayers. This self-assembly is dependent on the packing parameter of the lipid molecule. Packing parameter is defined as the ratio of the volume occupied by the hydrophobic tails per molecule 푣 (v) to the product of the head group area (a) and the lipid tail length (l), i.e., . Commonly used 푎푙
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