Distinguishing between the molecular interactions of synaptotagmin with SNAREs and with lipid bilayer using atomic force microscopy in recognition imaging mode

抄録

Synaptic vesicle fusion with the presynaptic plasma membrane is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins. Assembly of a complex between a vesicle-associated membrane protein, VAMP (a v-SNARE), in the vesicle membrane and a syntaxin 1A-SNAP-25 (t-SNAREs) heterodimer in the target presynaptic membrane drives fusion. Additionally, synaptotagmin acts as a Ca²⁺-sensitive and phosphatidylserine (PS)-dependent trigger protein to initiate fusion. To dissect out the interaction of synaptotagmin with the t-SNAREs, we used atomic force microscopy (AFM) in recognition imaging mode to study its binding to the syntaxin 1A-SNAP-25 complex integrated into lipid bilayers without PS. Using a synaptotagmin-functionalized AFM cantilever, we identified a Ca²⁺-independent binding signal between synaptotagmin and the syntaxin 1A-SNAP-25 complex at the single-molecule level. Within the nerve terminal, this Ca²⁺-independent interaction could potentially locate synaptotagmin close to the t-SNAREs in preparation for recognition of the incoming Ca²⁺ signal.