Vesicle fusion mediated by hybridization of transmembrane-anchored DNA [electronic resource]

Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. The process of vesicle fusion is often mediated by the SNARE proteins, which come together to form a four-helix bundle that brings the vesicle and target membrane into close proximity (docking), followed by merging of the outer leaflets to form a continuous bilayer (hemi-fusion) and eventual merging of the inner leaflets and content release (full fusion). Our system uses the hybridization of complementary DNA strands to model the formation of the SNARE four-helix bundle. Incoming vesicles and the target tethered bilayer each contain a DNA sequence anchored in the membrane by a single leaflet-spanning glycerol diether lipid. Hybridization of the two strands in a "zippering" orientation brings the membranes into close proximity, allowing fusion to occur. However, 70% of observed fusion events in the DNA-lipid system are arrested at the hemi-fusion stage, while only 5% eventually go to full fusion. This may be because the DNA-lipid spans only half the bilayer: upon hemi-fusion and mixing of the outer leaflets, the DNA-lipid is free to diffuse into the target membrane and away from the vesicle. This work expands upon our previous use of DNA-lipids to model vesicle fusion and investigates the influence of a transmembrane anchor on fusion outcomes. Unlike a single-leaflet anchor, a transmembrane anchor would be unable to diffuse away from the vesicle upon hemi-fusion and may thus increase the amount of eventual full fusion observed. There is conflicting evidence in the literature about the role of the transmembrane domain in SNARE-mediated fusion, whether it serves a passive role in the process or is necessary for fusion to occur. Our model system based on DNA hybridization allows us to study the effects of a transmembrane anchor in a simpler, more controlled system than one based on SNARE proteins. Initial investigations focused on a 32-amino acid peptide containing the C-terminal transmembrane domain of the SNARE protein synaptobrevin as a possible transmembrane anchor. Numerous attempts were made to synthesize the DNA-peptide via azide-alkyne and thiol linkages. Though characterization data indicated the successful formation of DNA-peptide product, low reaction yield and difficulty of quantitative purification hindered attempts to use the conjugate in our system. Subsequent research focused on the use of the polyisoprenoid solanesol as a transmembrane anchor. The solanesol thiol was synthesized and conjugated to thiol-DNA via disulfide linkage. DNA-solanesol was then incorporated into vesicles and the effect of a transmembrane lipid anchor on vesicle fusion investigated. When the solanesol anchor was present on the incoming vesicles, target membrane, or both, approximately twice as much full fusion was observed as in the DNA-lipid mediated system, as measured by lipid mixing. Similarly, content transfer experiments also indicated an increase in full fusion when one or both membranes contained DNA-solanesol. Even with this transmembrane anchor, full fusion with the target membrane totals at most 15% of all fusion events. While our DNA-lipid model system has previously indicated that vesicle fusion can occur in the absence of a transmembrane anchor, hemi-fusion was the predominant outcome and full fusion was rarely observed. In comparison, fusion mediated by DNA-solanesol more often leads to eventual full fusion, though these events are still in the minority. These results indicate that though a transmembrane anchor may increase the efficiency of full fusion, there may be other factors involved in the SNARE-mediated system that influence the outcomes of the fusion process.

Keywords: Chemistry.
Advisor: Boxer Steven G. (Steven George)
Burns Noah
Cegelski Lynette advisor. Thesis advisor