Optical Fusion Assay Based on Membrane-Coated Beads in a 2D Assembly

by Chunxiao Bao
Doctoral thesis
Date of Examination:2014-04-02
Date of issue:2014-04-25
Advisor:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Philipp Vana
Persistent Address: http://dx.doi.org/10.53846/goediss-4478

 

 

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Abstract

English

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
 
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