Cryo-electron Microscopy of Reconstituted Membrane Fusion Systems

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are responsible for driving the fusion of biological membranes and are present across many different cell trafficiking systems. Synaptic SNARE proteins allow the release of neurotransmitters across synaptic clefts by promoting the fusion of vesicle membranes with neuron plasma membranes. In doing so, neuron action potentials are successfully relayed from one neuron to the next. The process of vesicle fusion is achieved through the folding of vesicle v-SNARE proteins with plasma membrane t-SNARE proteins, which leads to the enthalpically driven merging of vesicle and plasma membranes. In order to ensure nerve transmission is regulated in a timely manner, a range of SNARE regulatory proteins exist so as to coordinate vesicle fusion with the arrival of an action potential. Though the biochemical action of SNAREs and their regulatory proteins have been well characterised, exactly how these proteins organise at synaptic vesicle fusion sites and therefore coordinate fusion is poorly understood. Through the usage of a SNARE reconstituted in-vitro system, I use cryo-electron tomography to provide insight into how SNARE proteins organise at fusion sites showing that such organisation occurs in a vesicle-plasma membrane distance-dependent manner. In order to improve the quality of structural data acquired at fusion sites and pursue a structural averaging approach, I later go on to investigate the design and implementation of alternative SNARE reconstituted systems. Among these, I develop methods by which cryo-EM grids can be coated with t-SNARE reconstituted bilayers. These bilayer-coated EM grids present a new method by which vesicle fusion may be studied and they also may also act as a new research tool by which other membrane proteins can be studied by cryo-EM.