Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology



First Advisor

Yeon-Kyun Shin


SNARE-mediated Ca2+ triggered membrane fusion is essential for neuronal communication. The speed at which this process is orchestrated is further emphasized because it serves as a temporal limit for cognitive and physical activities. However, attempts to recapitulate SNARE-mediated membrane fusion with SNAREs and synaptotagmin 1 (Syt1) between two single proteoliposomes came short in respect to Ca2+ sensitivity, speed and fusion efficiency compared to in vivo observations. This discrepancy raises concerns if there are some critical protein machinery that are missing or if the topology of the proteoliposome fusion assay does not faithfully represent synaptic vesicle and plasma membrane. Some suspect that the discrepancy might be due to the tight membrane curvature of proteoliposomes which may not mimic the relaxed curvature of the plasma membrane well. Others wonder if our long-standing dogma that SNAREs are the core membrane fusion machinery is valid.

In this study we investigate the role of complexin (Cpx) in a well-defined in vitro environment. Specifically, we observed Ca2+-triggered SNARE-mediated content-mixing between two proteoliposome pairs with total internal reflection (TIR) microscopy. We find that Cpx enhances Ca2+-triggered vesicle fusion with the yield changing from approximately 10% to 70% upon increasing Cpx from 0 to 100 nM. Unexpectedly, however, the fusion efficiency becomes reduced when Cpx is increased further, dropping to 20% in the µM range, revealing a bell-shaped dose-response curve.

With our Cpx assisted in vitro single vesicle-to-vesicle fusion assay which has high efficiency and physiologically relevant Ca2+ sensitivity, we investigated the inhibitory of effects botulinum toxins (BoNT) A and E. BoNT A and E both block neurotransmitter release by specifically cleaving the C-terminal ends of SNAP-25, a plasma membrane SNARE protein. Here, we find that SNAP-25A and E, the cleavage products of BoNT A and E respectively, terminate membrane fusion via completely different mechanisms. Specifically, SNAP-25E halts membrane fusion prior to the docking stage due to its incapability to support SNARE pairing. In contrast, SNAP-25A leads the fusion pathway faithfully prior to the fusion pore opening. The EPR results show that the discrepancy between SNAP-25A and E might stem from the extent of the dynamic destabilization of the t-SNARE core at the N-terminal half which plays a pivotal role in nucleating SNARE complex formation. Thus, the results provide insights into the structure and dynamics-based mechanism whereby BoNT A and E impairs membrane fusion.

While we observed the increase of Ca2+ sensitivity and fusion probability by incorporating Cpx into the in vitro single vesicle-to-vesicle fusion assay, we still were not able to observe fast synchronous fusion. Thus, we expand our investigation and probe the proteoliposome-to-supported bilayer fusion assay. We find that SNAREs, Syt1 and Ca2+ together can elicit more than a 50-fold increase in the number of membrane fusion events. What is more remarkable is that the docking-to-fusion delay of ~55% of all vesicle fusion occurs resides within 20 msec. Furthermore, Syt1 binding to t-SNAREs prior to Ca2+ inhibits spontaneous fusion leading to a loss of subsequent Ca2+ response. Thus, we propose a productive and non-productive pathway for Syt1 in which pre-binding of Ca2+ may be required for the productive pathway. We believe that the improved membrane fusion assay provides unprecedented opportunities to test the mechanistic models for Ca2+-triggered exocytosis in a time scale ever closer to the natural one.


Copyright Owner

Jaewook Kim



File Format


File Size

105 pages

Included in

Biophysics Commons