1. P-1 Genetically controlled fusion, exocytosis and fission of artificial vesicles E. Boenzli 1, M. Hadorn 1, D. de Lucrezia 2, M. G. Jørgensen 3, P. Eggenberger Hotz 4, M. M. Hanczyc 1, T. Yomo 5 References (1) Noireaux et al. A vesicle bioreactor as a step toward an artificial cell assembly. 2004. Proc Natl Acad Sci U S A 101(51): p. 17669-17674; (2) Caschera et al. Programmed Vesicle Fusion Triggers Gene Expression. 2011. Langmuir. In print; (3) Nomura et al. Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion. 2004. Proc Natl Acad Sci U S A. 101(10): p. 3420-5; (4) Hadorn and Eggenberger Hotz. Encapsulated Multi-vesicle Assemblies of Programmable Architecture: Towards Personalized Healthcare. 2011. CCIS. Heidelberg, Springer; (5) Pautot et al. Engineering asymmetric vesicles. 2003. Proc Natl Acad Sci U S A. 100(19): p. 10718-21; (6) Noireaux et al. Development of an artificial cell, from self-organization to computation and self-reproduction. 2011. Proc Natl Acad Sci U S A. 108(9): p. 3473-80; (7) Hadorn and Eggenberger Hotz. Towards Personalized Drug Delivery: Preparation of an Encapsulated Multicompartment System. 2010. BIOSTEC. Valencia, Spain. Acknowledgements We thank Steen Rasmussen (FLinT, SDU) and Martin Røssel Larsen (BMB, SDU), for their helpful contribution and support. Introduction Artificial vesicles represent ideal candidates as a model for artificial cells. It was shown that artificial and natural genetic programs and the required cellular machinery (cell-free expression systems) can be incorporated into vesicles and allow the synthesis of proteins 1, 2. In a second strand of research, vesicles were shown to fuse if a special class of viral proteins termed fusion peptides, were added to the external medium 3. In the present work, we intend to combine these two strands of research and to developed a scenario how a genetically controlled fission and exocytosis of vesicles may be achieved by the synthesis of fusion peptides within vesicles. Discussion The aim is to induce in a next step fusion, fission and exocytosis of vesicles (Fig. 3). Later, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. Changes in the pH are reported to control the activity of the fusion peptides 3. This project may present an important step towards an artificial cell. Especially vesicle fission is discussed as one of the upcoming challenges in designing an artificial cell 6. Moreover, it may become important in personalized drug delivery 7. Figure 3 Results We already reported a protocol 4 to produce nested vesicles. Here, we introduced fusion peptides (12.5-50 μM) (Fig. 1, a). The membranes of enclosed vesicles were also labeled with carboxyfluorescin (Fig. 1, b). Moreover, we enclosed a commercially available cell-free system in vesicles (99% POPC, 1% bPEG2000-DSPE) and expressed eGFP as a proof of principle (Fig. 1, c). Fig. 3. Principle of vesicle (a) exocytosis and (b) fission induced by amphipatic fusion peptides (red/green). The integration of the viral peptides to the inner leaflet of vesicles followed by peptide activation, e. g. by the pH 3, may destabilize the vesicle membrane and induce fusion, fission and exocytosis. ab a b c Fig. 1. Nested vesicles (a, b) and eGFP expression (c) in vesicles. Objective and bar: for all pictures the same as in c. Materials & Methods Figure 1 Figure 2 Fig. 2. The nested vesicle preparation protocol 4, modified from the water-in-oil emulsion transfer method 5, offers perfect control over the composition of both the bulk solution and the intravesicular solution. (1) Center for Fundamental Living Technology (FLinT), Department of Physics and Chemistry, SDU, Denmark; (2) European Centre for Living Technology, Ca’ Foscari University of Venice, Italy; (3) Department of Biochemistry and Molecular Biology (BMB), SDU, Denmark; (4) The Mærsk Mc-Kinney Møller Institute, SDU, Denmark; (5) Department of Bioinformatics Engineering, Osaka University, Japan a roadmap _