Optical Fusion Assay Based on Membrane-Coated Beads in a 2D Assembly
Bao, Chunxiao
Dissertation
Angenommen am:
2014-04-02
Erschienen:
2014-04-25
Betreuer:
Janshoff, Andreas Prof. Dr.
Gutachter:
Janshoff, Andreas Prof. Dr.
Gutachter:
Vana, Philipp Prof. Dr.
Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0022-5E98-A
Dateien
Name:
Chunxiao Bao_Thesis.pdf
Größe:
6,3 MB
Format:
PDF
Lizenzbestimmungen:
Zusammenfassung
Englisch
A novel assay of membrane fusion is established based on membrane-coated beads in a 2D assembly. This assay allows identifying the different stages of the fusion process. Membrane fusion driven by heterodimeric coiled coil formation as a proof of concept using fusogenic K- and E-peptides provided results comparable to those obtained with conventional liposome assays, but with additional information on docking efficiency. In this fusion assay, LBs and SBs are modified with i-K3Cys (LB-i-K3) and i-E3Cys (SB-i-E3) respectively and LBs are fluorescent labeled. The populations of fusogenic beads can be distinguished by size discrimination. When LB-i-K3and SB-i-E3 are mixed in water, the fusion process terminates in hemifusion. Interestingly, all fusion events including docking, hemifusion and full fusion are observed in the presence of Ca2+ in one experiment. This may due to the bridging effect of calcium ions binding to PC and non-reacted negative MCC-DOPE. Block or change the zipping conformational of coiled coil structure is necessary for the viral infection of host cells. In this bead-based assay, the introduction of inhibitors (i-E3Cys) efficiently decreases the hemifusion efficiency and abolishes full fusion (in the presence of Ca2+). Therefore, this approach is expected to be an invaluable tool to identify small-molecule inhibitors of viral fusion with unprecedented accuracy in prefusion state. Multiple fusion could be observed and distinguished, which takes place among more than two fusogenic membrane-coated beads. The orientations of coiled coil formation, including parallel and antiparallel orientations, are shown to have no effect on the fusion process in this bead-based assay. Most interestingly, size-dependent assays employed beads with increasing diameter scale, demonstrate that van der Waals interaction could provided energy for supported membrane detaching from the silica beads, hence promote membrane fusion.
Keywords: membrane fusion; membrane-coated beads
