Single-molecule analysis of synaptic protein complexes and vesicle recruitment
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Single-molecule pull-down (SIM-Pull) combined with TIRF microscopy enables direct visualization of proteins and multi-protein complexes. Here, we present an extended SIM-Pull protocol for analyzing protein interactions at the active zone and their ability to recruit isolated synaptic vesicles (SV). SV recruitment mediated by STX1A-SNARE or RIM1-Rab3a interactions, respectively; can be directly visualized and quantified. This technique opens new avenues to examine the subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context.
Highlights
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Extended SIM-Pull protocol combining biochemical isolation with TIRF microscopy to study synaptic protein complexes at near-native environment
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Enables direct quantification of synaptic vesicle recruitment at the surface (active zone) via protein-mediated vesicle tethering
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Adaptable platform for probing molecular interactions of protein complexes within different neuronal, cellular or subcellular compartments
Institutional permissions
Animals were handled and maintained according to the guidelines laid down by the Animal Welfare Body (AWB) (Instantie voor Dierenwelzijn IvD) in line with the animal experimentation policy within Radboud University and RadboudUMC; under the license/protocol numbers 2021-0040-001/002 to Dr. Anne-Sophie Hafner.
