Abstract
In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.
Highlights
Neurons are the foundation for many fundamental processes in the human body
The vesicles and the supported bilayer have been reconstituted with VAMP2 and t-SNAREs (Syntaxin-1A and SNAP-25), respectively
When we repeated the assay under the SNARE only conditions, we found that out of 170 events, 47% (80 events) advanced to a large pore state, while 53% (90 events) did not develop the large fusion pore
Summary
Neurons are the foundation for many fundamental processes in the human body. The central nervous system governs movement, sensory, cognition, and memory. The neurons that compose the nervous system must be able to communicate with each other in a highly regulated manner to facilitate the exquisite orchestration necessary for these essential processes. They execute this via neurotransmitter release into the synapse. The pre-synapse engages in an important mechanism called vesicle fusion in which vesicles, containing neurotransmitters, fuse with the plasma membrane. This fusion allows neurotransmitter release into the synaptic cleft so that they can transmit a signal from one neuron to the via binding to receptors on the post synaptic membrane.
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