Lipid Vesicle Fusion: Investigation, Generation and Manipulation of Cell-Membrane Mimics
Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. In this work, cell-membrane mimics were constructed and evaluated as models for studies of the membrane-fusion process. As a mimic of SNARE-proteins, known to induce fusion in secretory cells (exocytosis), short cholesterol-tagged DNA strands were used to facilitate fusion. The DNA strands were proven efficient as SNARE-protein substitutes in terms of bringing lipid vesicle fusion and content release about. This thus enabled reductionist and protein-free studies of some of the mechanisms behind lipid vesicle fusion. In particular, the fast kinetics of vesicle-content release upon fusion was possible to resolve using amperometry. The model system designed to be compatible with amperometric recordings generated data that could be directly compared with amperometric recordings from live cells. In combination with theoretical modeling, this has led us to suggest that the opening of the fusion pore is the rate-limiting step for content release in a protein-free system. In combination with the ability to systematically alter additional parameters, such as lipid composition, this model system may help resolving some of the still unanswered questions regarding the molecular mechanisms that control the exocytosis process.
As a second approach, lipid vesicle fusion was used as a new means to form planar cell-membrane mimics on solid supports. It is demonstrated that such supported lipid bilayers (SLBs) could be made with complex lipid compositions that are generally prevented when alternative SLB-formation methods are used. In particular, successful formation of SLBs containing native cell membrane components was demonstrated. Furthermore, by forming such SLBs in microfluidic systems, spatial manipulation of cell-membrane-associated molecules was demonstrated. This, in turn, points towards the exciting possibility to both enrich and separate membrane proteins while in their native environment, potentially offering a new way to study this biologically and pharmacologically very important class of biomolecules.
fluorescence resonance energy transfer
spreading lipid bilayers
total internal reflection fluorescence microscopy