Access Type

Dissertation/Thesis

Date of Award

January 2023

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

Maria Bykhovskaia

Abstract

Neurons communicate via releasing neurotransmitters from fused synaptic vesicles (SV). The fusion of SVs can occur spontaneously, which is implicated in synaptic development and synaptic homeostatic plasticity. Vesicle fusion is mediated by the core fusion machinery, including the SNARE complex: a four helical bundle which attaches the SV to the plasma membrane (PM). The SNARE complex zippers together into a coil coiled bundle to initiate fusion. Complexin (Cpx), a cytosolic protein, regulates spontaneous vesicle fusion, but the exact clamping mechanism remains unclear. Our lab previously proposed the “Late Clamp” model which hypothesizes that Cpx clamps spontaneous fusion by stabilizing a SNARE-Cpx state where the C-terminus of synaptobrevin (Syb), the vesicle-attached SNARE protein, is partly separated (approximately 2-3 layers or helical turns) from the complex, interacting with the Cpx AH. In this study, this model was further developed and verified in Drosophila through botulinum neurotoxin (BoNT) loading, molecular modeling, electrophysiology, and Cpx mutagenesis. For loading, serotypes BoNT/B and BoNT/G were chosen because they cut Syb at different locations: BoNT/G cleaving around Layer 7 while BoNT/B cleaves more distally around Layer 6. Loading these serotypes revealed a SNARE-Cpx pre-fusion complex where Syb is partially unraveled by 2-3 helical turns (Layers 7-9), validating the clamped state of the “Late Clamp” model. Further supporting the model, Cpx mutagenesis demonstrated that the Cpx AH is essential for clamping. Modeling the “Late Clamp” SNARE-Cpx complex between lipid bilayers revealed that the Cpx AH stabilizes the clamp via interactions with the partially unraveled Syb C-terminus as well as with SV bilayer lipids. This finding that the Cpx AH stabilizes clamping by also interacting with the SV bilayer further developed the model. Most importantly, the model guided Cpx site-directed mutagenesis and was validated by successfully predicting spontaneous fusion phenotypes (one poor-clamping mutant and one super-clamping mutant) of the mutants in both molecular simulations and in vivo. In total, the results support a developed “Late Clamp” model where the Cpx AH clamps fusion by interacting with the partially unraveled C-terminus of Syb (2-3 helical turns or layers) and interacts with the SV bilayer to further stabilize this state.

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